Synthetic molecule constructs

ABSTRACT

Synthetic molecule construct of the structure F-S1-S2-L where F—S1 is an aminoalkylglycoside and S2-L is a dicarboxylated, e.g. adipylated, phosphatidylethanolamide are disclosed. The two terminal sugars of the aminoalkylglycoside (Galα1-3Galβ-) are the two terminal sugars of the Galili antigen.

This application is a continuation-in-part (CIP) of U.S. application Ser. No. 15/804,427 filed Nov. 6, 2017, which is a CIP of 15/168,144 filed May 30, 2016 (U.S. Pat. No. 9,809,614), which is a continuation of 14/108,749 filed Dec. 17, 2013 (U.S. Pat. No. 9,353,349), which is a continuation of 13/067,021 filed May 3, 2011 (U.S. Pat. No. 8,637,473), which is a divisional of 10/593,829 filed Jan. 12, 2007 (U.S. Pat. No. 8,013,131), which is the U.S. national phase of PCT/NZ2005/000052 filed Mar. 22, 2005, which claims priority to New Zealand Patent Application Nos. 531866 filed Mar. 22, 2004, and 537941 filed Jan. 28, 2005, the contents of each of which are hereby incorporated by reference. 15/804,427 is also a CIP of 14/972,301 filed Dec. 17, 2015 which is a CIP of 14/085,156 filed Nov. 20, 2013 (U.S Pat. No. 9,226,968), which is a continuation of 13/459,399 filed Apr. 30, 2012 (abandoned), which is a continuation of 12/451,120 filed Mar. 29, 2010 (U.S. Pat. No. 8,211,860), which is the U.S. national phase of PCT/NZ2008/000095 filed Apr. 28, 2008, the contents of each of which are hereby incorporated by reference.

This application is also a CIP of 15/279,652 filed Sep. 29, 2016, which is a continuation of 14/563,127 filed Dec. 8, 2014, which is a continuation of 13/354,449 filed Jan. 20, 2012 (abandoned), which is a continuation of 12/310,803 filed Mar. 4, 2010 (abandoned), which is the U.S. national phase of PCT/NZ2007/000256 filed Sep. 6, 2007, which claims priority to New Zealand Patent Application Nos. 549742 filed Sep. 6, 2006, and 549740 filed Sep. 7, 2006, the contents of each of which are hereby incorporated by reference.

FIELD OF INVENTION

The invention relates to synthetic molecules that spontaneously and stably incorporate into lipid bilayers, including cell membranes. Particularly, although not exclusively, the invention relates to the use of these constructs to effect qualitative and quantitative changes in the expression of antigens and flurorophores at the surface of cells.

BACKGROUND

Cell surface antigens mediate a range of interactions between cells and their environment. These interactions include cell-cell interactions, cell-surface interactions and cell-solute interactions. Cell surface antigens also mediate intra-cellular signalling.

Cells are characterised by qualitative and quantitative differences in the cell surface antigens expressed. Qualitative and quantitative changes in the cell surface antigens expressed alter both cell function (mode of action) and cell functionality (action served).

The marking of cells by conjugation of a fluorophore with a surface expressed antigen may affect cell function. Furthermore, mobility of the fluorophore within the two dimensions of the cell membrane is necessarily dependent on the mobility of the conjugated antigen.

Being able to localise fluorophores to the surface of cells without these limitations or effect qualitative and/or quantitative changes in the surface antigens expressed by a cell has diagnostic and therapeutic value. Transgenic and non-transgenic methods of effecting qualitative and/or quantitative changes in the surface antigens expressed by a cell are known.

Protein painting is a non-transgenic method for effecting qualitative and/or quantitative changes in the surface antigens expressed by a cell. The method exploits the ability of GPI linked proteins to spontaneously anchor to the cell membrane via their lipid tails. The method described in the specification accompanying international application no. PCT/US98/15124 (publ. no. WO 99/05255) includes the step of inserting a GPI linked protein isolated from a biological source into a membrane. Isolated GPI-anchored proteins are stated as having an unusual capacity to reintegrate with a cell-surface membrane.

Cells exist in an aqueous environment. The cell membrane is a lipid bilayer that serves as a semi-permeable barrier between the cytoplasm of the cell and this aqueous environment. Localising antigens to the cell surface may also be achieved by the use of glycolipids as membrane anchors.

The method described in the specification accompanying international application no. PCT/NZ02/00214 (publ. no. WO 03/034074) includes the step of inserting a controlled amount of glycolipid into a membrane. The amount of glycolipid inserted is controlled to provide cells with a desired level of antigen expression.

The method described in the specification accompanying international application no. PCT/NZ03/00059 (publ. no. WO 03/087346) includes the step of inserting a modified glycolipid into a membrane as a “membrane anchor”. The modified glycolipid provides for the localisation of antigens to the surface of the cell or multicellular structure. New characteristics may thereby be imparted on the cell or multicellular structure.

These methods typically include the isolation of a glycolipid or glycolipid-linked antigen from a biological source. The isolation of glycolipids or glycolipid-linked antigens from biological sources is costly, variable and isolatable amounts are often limited. Obtaining reagents from zoological sources for therapeutic use is particularly problematic, especially where the reagent or its derivative products are to be administered to a human subject.

Synthetic molecules for which the risk of contamination with zoo-pathogenic agents can be excluded are preferred. Synthetic counterparts for naturally occurring glycolipids and synthetic neo-glycolipids have been reported. However, for a synthetic glycolipid to be of use as a membrane anchor it must be able to spontaneously and stably incorporate into a lipid bi-layer from an aqueous environment. The utility of synthetic glycolipids in diagnostic or therapeutic applications is further limited to those synthetic glycolipids that will form a solution in saline.

Organic solvents and/or detergents used to facilitate the solubilization of glycolipids in saline must be biocompatible. Solvents and detergents must often be excluded or quickly removed as they can be damaging to some cell membranes. The removal of solvents or detergents from such preparations can be problematic.

Damage to cell membranes is to be avoided especially where the supply of cells or multicellular structures is limited, e.g. embryos, or the cells are particularly sensitive to perturbation, e.g. hepatocytes.

Methods of localising fluorophores and antigen to the surface of cells that avoid affecting cell function and provide for independent mobility of the fluorophore or antugen within the two dimensions of the cell membrane are desired.

There exists a need for water soluble synthetic molecules that can be used to localise fluorophores to the surface of cells or are functionally equivalent to naturally occurring glycolipids and glycolipid-linked antigens in respect of their ability to spontaneously and stably incorporate into lipid bi-layers, including cell membranes.

Providing such synthetic molecules would obviate the limitations of marking cells by conjugation of a fluorophore with a surface expressed antigen. Providing such synthetic molecules would also obviate the limitations of glycolipids and glycolipid-linked antigens isolated from biological sources and facilitate being able to effect qualitative and/or quantitative changes in the surface antigens expressed by a cell.

It is an object of this invention to provide such synthetic molecules and a method for their preparation. It is a further object of this invention to provide synthetic molecules for use in diagnostic and therapeutic applications. The preceding objects are to be read disjunctively with the object to at least provide the public with a useful choice.

STATEMENTS OF INVENTION

In a first aspect the invention provides a synthetic molecule construct of the structure F—S₁-S₂-L where:

-   -   F is selected from the group consisting of carbohydrates and         fluorophores;     -   S₁-S₂ is a spacer linking F to L; and     -   L is a lipid selected from the group consisting of diacyl- and         dialkyl-glycerolipids, including glycerophospholipids, and         sphingosine derived diacyl- and dialkyl-lipids, including         ceramide.

Preferably, L is a lipid selected from the group consisting of diacyl- and dialkyl-glycerolipids, including glycerophospholipids. More preferably, L is selected from the group consisting of: diacylglycerolipids, phosphatidate, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine, phosphatidyl inositol, phosphatidyl glycerol, and diphosphatidyl glycerol derived from one or more of trans-3-hexadecenoic acid, cis-5-hexadecenoic acid, cis-7-hexadecenoic acid, cis-9-hexadecenoic acid, cis-6-octadecenoic acid, cis-9-octadecenoic acid, trans-9-octadecenoic acid, trans-11-octadecenoic acid, cis-11-octadecenoic acid, cis-11-eicosenoic acid or cis-13-docsenoic acid. More preferably, the lipid is derived from one or more cis-destaurated fatty acids. Most preferably, L is selected from the group consisting of: 1,2-O-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE), 1,2-O-distearyl-sn-glycero-3-phosphatidylethanolamine (DSPE) and rac-1,2-dioleoylglcerol (DOG).

Preferably, L is a glycerophospholipid and the molecule includes the substructure:

where n is 3 to 5, X is H or C, and

is other than H. Preferably, n is 3.

Preferably, the molecule is water soluble.

Preferably, the molecule spontaneously incorporates into a lipid bi-layer when a solution of the molecule is contacted with the lipid bi-layer. More preferably, the molecule stably incorporates into the lipid bilayer.

Preferably, F, S_(1,) S₂ and L are covalently linked.

S₁-S₂ is selected to provide a water-soluble synthetic molecule construct.

In a first embodiment F is a naturally occurring or synthetic glycotope. Preferably, F is a naturally occurring or synthetic glycotope consisting of three (trisaccharide) or more sugar units. More preferably, F is a glycotope selected from the group consisting of lacto-neo-tetraosyl, lactotetraosyl, lacto-nor-hexaosyl, lacto-iso-octaosyl, globoteraosyl, globo-neo-tetraosyl, globopentaosyl, gangliotetraosyl, gangliotriaosyl, gangliopentaosyl, isoglobotriaosyl, isoglobotetraosyl, mucotriaosyl and mucotetraosyl series of oligosaccharides. Most preferably, F is selected from the group of glycotopes comprising the terminal sugars: GalNAcα1-3(Fucα1-2)Galβ; Galα1-3Galβ; Galβ; Galα1-3(Fucα1-2)Galβ; NeuAcα2-3Galβ; NeuAcα2-6Galβ; Fucα1-2Galβ; Galα1-4GlcNAcβ1-6(Galβp1-4GlcNAcβ1-3)Galβ; Fucα1-2Galβ1-4GlcNAcβ1-6(Fucα1-2Galβ1-4GlcNAcβ1-3)Galβ; Fucα1-2Galβ1-4GlcNAcβ1-6(NeuAcβ2-3Galβ1-4GlcNAcβ1-3)Galβ; NeuAcα2-3Galβ1-4GlcNAcβ1-6(NeuAcα2-3Galβ1-4GlcNAβ1-3)Galβ; Galα1-4Galβ1-4Glc; GalNAcβ1-3Galα1-4Galβ1-4Glc; GalNAcα1-3GalNAcβ1-3Galα1-4Galβ1-4Glc; or GalNAcβ1-3GalNAcβ1-3Galα1-4Galβ1-4Glc.

When F is a glycotope, L is a glycerophospholipid and S₂ is selected from the group including: —CO(CH₂)₃CO—, —CO(CH₂)₄CC— (adipate), —CO(CH₂)₅CO— and —CO (CH₂)₅NHCO(CH₂)₅Co—, preferably S₁ is a C₃₋₅-aminoalkyl selected from the group consisting of: 3-aminopropyl, 4-aminobutyl, or 5-aminopentyl. More preferably S₁ is 3-aminopropyl.

In a second embodiment F is a molecule that mediates a cell-cell or cell-surface interaction. Preferably F is a carbohydrate with an affinity for a component expressed on a targeted cell or surface. More preferably F has an affinity for a component expressed on epithelial cells or extra-cellular matrices. Yet more preferably F has an affinity for a component expressed on the epithelial cells or the extra-cellular matrix of the endometrium. Most preferably the component expressed on the epithelial cells or the extra-cellular matrix of the endometrium can be a naturally expressed component or an exogenously incorporated component.

In a third embodiment F is a molecule that mediates a cell-solute interaction. Preferably F is a ligand for a binding molecule where the presence of the binding molecule is diagnostic for a pathological condition. More preferably F is a ligand for an antibody (immunoglobulin).

In a fourth embodiment F is a fluorophore. Preferably, F is a fluorophore selected from the group consisting of: fluorophores of fluorescein, Oregon

Green, Pennsylvania Green, Tokyo Green, eosin, BODIPY, BODIPY TR, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 488, Alexa Fluor 568, Alexa Fluor 594, Texas Red, Lucifer Yellow, tetramethylrhodamine and their derivatives. Most preferably, F is selected from the group consisting of: fluorophores of fluorescein, BODIPY and their derivatives.

Preferably, where F is the fluorophore of fluorescein or one of its derivatives, Si is a C₃₋₅-diaminoalkyl derivative selected from the group consisting of: 1,3-diaminopropyl, 1,4-diaminobutyl, or 1,5-aminopentyl derivatives. More preferably, where F is the fluorophore of fluorescein or one of its derivatives, S₁ is a C₃₋₅-aminoalkylthioureidyl. Most preferably, where F is the fluorophore of fluorescein or one of its derivatives, S₁ is 5-((5-aminopentyl) thioureidyl.

Preferably, where F is the fluorophore of fluorescein or one of its derivatives, S₂ is selected from the group including: —CO(CH₂)₃CO—, —CO(CH₂)₄CO—, —CO(CH₂)₅CO—and —CO(CH₂)₅NHCO(CH₂)₅CO—. More preferably, where F is the fluorophore of fluorescein or one of its derivatives, S₂ is —CO(CH₂)₄CO—.

Preferably, where F is the fluorophore of fluorescein or one of its derivatives, the structure includes the substructure:

where m and n are independently 3 to 5 and

is other than H.

Preferably, where F is the fluorophore of BODIPY or one of its derivatives, S₁ is a C₃₋₅-alkionyldiamine. More preferably, where F is the fluorophore of BODIPY or one of its derivatives, S₁ is propionyl ethyldiamine.

Preferably, where F is the fluorophore of BODIPY or one of its derivatives, S₂ is selected from the group consiting of: —CO(CH₂)₃CO—, —CO(CH₂)₄CO— and —CO(CH₂)₅CO—. More preferably, where F is the fluorophore of BODIPY or one of its derivatives, S₂ is —CO(CH₂)₄CO—.

Preferably, where F is the fluorophore of BODIPY or one of its derivatives the structure includes the substructure:

where p, q and r are independently 3 to 5 and * is other than H. More preferably, the sum of p, q and r is 8. Most preferably, p is 2, q is 2 and r is 4.

In specific embodiments the synthetic molecule construct has the structure:

the structure:

the structure:

the structure

the structure:

the structure:

the structure:

the structure:

the

structure:

the structure:

the structure:

the structure:

the structure:

the structure:

the structure:

or the structure:

M is typically H, but may be replaced by another monovalent cation such as Na⁺, K⁺ or NH₄ ⁺.

In a second aspect the invention provides a method of preparing a synthetic molecule construct of the structure F—S₁-S₂-L including the steps:

-   -   1. Reacting an activator (A) with a lipid (L) to provide an         activated lipid (A-L);     -   2. Derivatising an antigen (F) to provide a derivatised antigen         (F—S₁); and     -   3. Condensing A-L with F—S₁ to provide the molecule;

where:

-   -   A is an activator selected from the group including:         bis(N-hydroxysuccinimidyl), bis(4-nitrophenyl),         bis(pentafluorophenyl), bis(pentachlorophenyl) esters of         carbodioic acids (C₃ to C₇);     -   L is a lipid selected from the group consisting of diacyl- and         dialkyl-glycerolipids, including glycerophospholipids, and         sphingosine derived diacyl- and dialkyl-lipids, including         ceramide.     -   F is selected from the group consisting of carbohydrates; and     -   S₁-S₂ is a spacer linking F to L where S₁ is selected from the         group including: primary aminoalkyl, secondary aliphatic         aminoalkyl or primary aromatic amine; and S₂ is absent or         selected from the group including: —CO(CH₂)₃CO—, —CO(CH₂)₄CO—         (adipate), and —CO(CH₂)₅CO—.

Preferably, the molecule is water soluble.

Preferably, the molecule spontaneously incorporates into a lipid bi-layer when a solution of the molecule is contacted with the lipid bi-layer. More preferably the molecule stably incorporates into the lipid bilayer.

Preferably, F, S₁, S₂ and L are covalently linked.

Preferably, F is selected from the group consisting of naturally occurring or synthetic glycotopes.

Preferably, L is a lipid selected from the group consisting of diacyl- and dialkyl-glycerolipids, including glycerophospholipids. More preferably L is selected from the group consisting of: diacylglycerolipids, phosphatidate, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine, phosphatidyl inositol, phosphatidyl glycerol, and diphosphatidyl glycerol derived from one or more of trans-3-hexadecenoic acid, cis-5-hexadecenoic acid, cis-7-hexadecenoic acid, cis-9-hexadecenoic acid, cis-6-octadecenoic acid, cis-9-octadecenoic acid, trans-9-octadecenoic acid, trans-11-octadecenoic acid, cis-11-octadecenoic acid, cis-11-eicosenoic acid or cis-13-docsenoic acid. More preferably the lipid is derived from one or more cis-destaurated fatty acids. Most preferably L is selected from the group consisting of: 1,2-O-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE), 1,2-O-distearyl-sn-glycero-3-phosphatidylethanolamine (DSPE) and rac-1,2-dioleoylglycerol (DOG).

Preferably, L is a glycerophospholipid and the molecule includes the substructure:

where n=3 to 5, X is H or C, and

is other than H. Preferably n is 3.

Preferably, A (R-S₂) and Si are selected to provide a water soluble synthetic molecule construct.

In a first embodiment F is a naturally occurring or synthetic glycotope. Preferably, F is a naturally occurring or synthetic glycotope consisting of three (trisaccharide) or more sugar units. More preferably, F is a glycotope selected from the group consisting of lacto-neo-tetraosyl, lactotetraosyl, lacto-nor-hexaosyl, lacto-iso-octaosyl, globoteraosyl, globo-neo-tetraosyl, globopentaosyl, gangliotetraosyl, gangliotriaosyl, gangliopentaosyl, isoglobotriaosyl, isoglobotetraosyl, mucotriaosyl and mucotetraosyl series of oligosaccharides. Most preferably, F is selected from the group of glycotopes comprising the terminal sugars GalNAcα1-3(Fucα1-2)Galβ; Galα1-3Galβ; Galβ; Galα1-3(Fucαl-2)Galβ; NeuAcα2-3Galβ3; NeuAcα2-6Galβ; Fucα1-2Galβ1-4GlcNAcβ1-6(Galβ1-4GlcNAcβ1-3)Galβ; Fucα1-2Galβ1-4GlcNAcβ1-6(Fucα1-2Galβ1-4GlcNAcβ1-3)Galβ; Fucα1-2Galβ1-4GlcNAcβ1-6(NeuAcα2-3Galβ1-4GlcNAcβ1-3)Galβ; NeuAcα2-3Galβ1-4GlcNAcβ1-6(NeuAcα2-3Galβ1-4GlcNAcβ1-3)Galβ; Galα1-4Galβ1-4Glc; GalNAcβ1-3Galα1-4Galβ1-4Glc; GalNAcα1-3GalNAcβ1-3Galα1-4Galβ1-4Glc; or GalNAcβ1-3GalNAcβ1-3Galaα1-4Galpβ1-4Glc.

When F is a glycotope, L is a glycerophospholipid and S2 is selected from the group including: —CO(CH₂)₃CO—, —CO(CH₂)₄CO—, —CO(CH₂)₅CO— and —CO(CH₂)₅NHCO(CH₂)₅CO—. Preferably, S₁ is a C₃₋₅-aminoalkyl selected from the group consisting of: 3-aminopropyl, 4-aminobutyl, or 5-aminopentyl. More preferably, Si is 3-aminopropyl.

In a second embodiment F is a molecule that mediates a cell-cell or cell-surface surface interaction. Preferably, F is carbohydrate with an affinity for a component expressed on a targeted cell or surface. More preferably, F has an affinity for a component expressed on epithelial cells or extra-cellular matrices. Yet more preferably, F has an affinity for a component expressed on the epithelial cells or the extra-cellular matrix of the endometrium. Most preferably, the component expressed on the epithelial cells or the extra-cellular matrix of the endometrium can be a naturally expressed component or an exogenously incorporated component.

In a third embodiment F is a molecule that mediates a cell-solute interaction. Preferably F is a ligand for a binding molecule where the presence of the binding molecule is diagnostic for a pathological condition.

More preferably F is a ligand for an antibody (immunoglobulin).

In specific embodiments the water-soluble synthetic molecule construct has the structure:

the structure:

the structure:

the structure

the structure:

the structure:

the structure:

the structure:

or the structure:

M is typically H, but may be replaced by another monovalent cation such as Na⁺, K⁺ or NH₄ ⁺.

In a third aspect the invention provides a water-soluble synthetic molecule construct prepared by a method according to the second aspect of the invention.

In a fourth aspect the invention provides a method of effecting qualitative and/or quantitative changes in the surface antigens expressed by a cell or multi-cellular structure including the step of contacting a suspension of the cell or multi-cellular structure with a synthetic molecule construct according to the first aspect or third aspect of the invention for a time and at a temperature sufficient to effect the qualitative and/or quantitative change in the surface antigens expressed by the cell or multi-cellular structure.

Preferably, the cell or multi-cellular structure is of human or murine origin.

Preferably, the concentration of the water-soluble synthetic membrane anchor or synthetic molecule construct in the suspension is in the range 0.1 to 10 mg/mL.

Preferably, the temperature is in the range 2 to 37 ° C. More preferably the temperature is in the range 2 to 25 ° C. Most preferably, the temperature is in the range 2 to 4 ° C.

In a first embodiment the cell is a red blood cell.

In this embodiment, F is preferably selected from the group of glycotopes comprising the terminal sugars GalNAcα1-3(Fucαl-2)Galβ; Galα1-3Galβ; Galβ; Galα1-3(Fucα1-2)Galβ; NeuAcα2-3Galβ; NeuAcα2-6Galβ; Fucα1-2Galβ; Galβ1-4G1cNAcβ1-6(Galβ1-4G1cNAcβ1-3)Galβ; Fucα1-2Galα1-4GlcNAcβ1-6(Fucα1-2Galβ1-4G1cNAcβ1-3)Galβ; Fucα1-2Galβ1-4GlcNAcβ1-6(NeuAcα2-3Galβ1-4GlcNAcβ1-3)Galβ; NeuAcα2-3Galβ1-4GlcNAcβ1-6(NeuAcα2-3Galβ1-4GlcNAcβ1-3)Galβ; Galα1-4Galβ1-4Glc; GalNAcβ1-3Galα1-4Galβ1-4Glc; GalNAcα1-3GalNAcβ1-3Galα1-4Galβ1-4Glc; or GalNAcβ1-3GalNAcβ1-3Galα1-4Galβ1-4Glc. More preferably, F is selected from the group of glycotopes consisting of the oligosaccharides GalNAcα1-3(Fucα1-2)Galβ and Galα1-3(Fucα1-2)Galβ.

Preferably, the synthetic molecule construct is selected from the group including: A_(tri)-sp-Ad-DOPE (I); A_(tri)-spsp₁-Ad-DOPE (II); A_(tri)-sp-Ad-DSPE (III); B_(tri)-sp-Ad-DOPE (VI); H_(tri)-sp-Ad-DOPE (VII); H_(di)-sp-Ad-DOPE (VIII); Galβ_(i)-sp-Ad-DOPE (IX); Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAc-sp-Ad-DOPE (XII); and Fucαl-2Galβ1-3(Fucα1-4)GlcNAc-sp-Ad-DOPE (XIII).

In a second embodiment the multi-cellular structure is an embryo.

In this embodiment, F preferably is an attachment molecule where the attachment molecule has an affinity for a component expressed on the epithelial cells or the extra-cellular matrix of the endometrium.

The component expressed on the epithelial cells or the extra-cellular matrix of the endometrium can be a naturally expressed component or an exogenously incorporated component.

Preferably, the synthetic membrane anchor or synthetic molecule construct is selected from the group including: A_(tri)-sp-Ad-DOPE (I); A_(tri)-spspi-Ad-DOPE (II); A_(tri)-sp-Ad-DSPE (III); B_(tri)-sp-Ad-DOPE (VI); H_(tri)-sp-Ad-DOPE (VII); H_(di)-sp-Ad-DOPE (VIII); Galβ₁-sp-Ad-DOPE (IX); Fucαl-2Galβ1-3GlcNAβ1-3Galβ1-4GlcNAc-sp-Ad-DOPE (XII); and Fucαl-2Galβ1-3(Fucαl-4)GLcNAc-sp-Ad-DOPE (XIII).

In a third embodiment the cell is red blood cell.

In this embodiment, F is preferably a ligand for a binding molecule where the presence of the binding molecule is diagnostic for a pathological condition. More preferably, F is a ligand for an antibody (immunoglobulin).

In a fifth aspect the invention consists in a cell or multi-cellular structure incorporating a water-soluble synthetic molecule construct according to the first or third aspect of the invention.

Preferably, the cell or multi-cellular structure is of human or murine origin.

In a first embodiment the cell is a red blood cell incorporating a water soluble synthetic molecule construct selected from the group including: A_(tri)-sp-Ad-DOPE (I); A_(tri)-spsp₁-Ad-DOPE (II); A_(tri)-sp-Ad-DSPE (III); B_(tri)-sp-Ad-DOPE (VI); H_(tri)-sp-Ad-DOPE (VII); H_(di)-sp-Ad-DOPE (VIII); Galβ_(i)-sp-Ad-DOPE (IX); Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAc-sp-Ad-DOPE (XII); and Fucαl-2Galβ1-3(Fucα1-4)GlcNAc-sp-Ad-DOPE (XIII).

In a second embodiment the multi-cellular structure is an embryo incorporating a water soluble synthetic molecule construct selected from the group consisting of: A_(tri)-sp-Ad-DOPE (I); A_(tri)-spsp₁-Ad-DOPE (II); A_(tri)-sp-Ad-DSPE (III); B_(tri)-sp-Ad-DOPE (VI); H_(tri)-sp-Ad-DOPE (VII); H_(di)-sp-Ad-DOPE (VIII); Galβ_(i)-sp-Ad-DOPE (IX); Fucα1-2Galβ1-3GlcNAcβ1-3Gaβ1-4GlcNAc-sp-Ad-DOPE (XII); and Fucαl-2Galβ1-3(Fucα1-4)GlcNAc-sp-Ad-DOPE (XIII).

In a sixth aspect the invention consists in a kit comprising a dried preparation or solution of a water-soluble synthetic membrane anchor or synthetic molecule construct according to the first or third aspect of the invention.

Preferably, the water soluble synthetic molecule construct according to the first or third aspect of the invention is selected from the group consisting of: A_(tri)-sp-Ad-DOPE (I); A_(tri)-spsp₁-Ad-DOPE (II); A_(tri)-sp-Ad-DSPE (III); B_(tri)-sp-Ad-DOPE (VI); H_(tri)-sp-Ad-DOPE (VII); H_(di)-sp-Ad-DOPE (VIII); Galβ_(i)-sp-Ad-DOPE (IX); Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAc-sp-Ad-DOPE (XII); and Fucαl-2Galβ1-3(Fucαl-4)GlcNAc-sp-Ad-DOPE (XIII).

In a seventh aspect the invention consists in a kit comprising a suspension in a suspending solution of cells or multi-cellular structures according to the fifth aspect of the invention.

Preferably, the suspending solution is substantially free of lipid.

Preferably, the cell or multi-cellular structure is of human or murine origin.

Preferably, the cells are red blood cells that do not naturally express A- or B-antigen and incorporate a water soluble synthetic molecule construct selected from the group consisting of: A_(tri)-sp-Ad-DOPE (I); A_(tri)-spspi-Ad-DOPE (II); A_(tri)-sp-Ad-DSPE (III); B_(tri)-sp-Ad-DOPE (VI); H_(tri)-sp-Ad-DOPE (VII); H_(di)-sp-Ad-DOPE (VIII); Galβ₁-sp-Ad-DOPE (IX); Fucα1-2Galβ1-3GlcNAcβ1-3Galpβ1-4GlcNAc-sp-Ad-DOPE (XII); and Fucα1-2Galβ1-3(Fucα1-4)GlcNAc-sp-Ad-DOPE (XIII). More preferably, the cells are sensitivity controls.

In an eighth aspect the invention consists in a pharmaceutical preparation comprising a dried preparation or solution of a water-soluble synthetic molecule construct according to the first or fourth aspect of the invention.

Preferably, the pharmaceutical preparation is in a form for administration by inhalation.

Preferably, the pharmaceutical preparation is in a form for administration by injection.

In a ninth aspect the invention consists in a pharmaceutical preparation comprising cells or multi-cellular structures according to the fifth aspect of the invention.

Preferably, the cells or multi-cellular structures are of human or murine origin.

Preferably, the pharmaceutical preparation is in a form for administration by inhalation.

Preferably, the pharmaceutical preparation is in a form for administration by injection.

In a tenth aspect the invention provides a fluorescent cell marker of the structure:

F—S ₁-S ₂-L

including the substructure:

where

-   -   F is a fluorophore;     -   S₁-S₂ is a spacer linking F to L;     -   L is a lipid selected from the group consisting of diacyl- and         dialkyl-glycerolipids, including glycerophospholipids;     -   m and n are independently 3 to 6;     -   R₁is Oor S; and

-   -   is other than H.

The spacer (S₁-S₂) is selected to provide a water-soluble cell marker.

Preferably, F is selected from the group consisting of: fluorophores of fluorescein, Oregon Green, Pennsylvania Green, Tokyo Green, eosin, BODIPY, BODIPY TR, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 488, Alexa Fluor 568, Alexa Fluor 594, Texas Red, Lucifer Yellow, tetramethylrhodamine and their derivatives. Most preferably, F is selected from the group consisting of: fluorophores of fluorescein, BODIPY and their derivatives.

Preferably, the sum of m and n is 6 to 9 and * is C or N.

Preferably, where F is the fluorophore of fluorescein or one of its derivatives, S₁ is a C₃₋₅-diaminoalkyl derivative selected from the group consisting of: 1,3-diaminopropyl, 1,4-diaminobutyl, or 1,5-aminopentyl derivatives. More preferably, where F is the fluorophore of fluorescein or one of its derivatives, S₁ is a C₃₋₅-aminoalkylthioureidyl. Most preferably, where F is the fluorophore of fluorescein or one of its derivatives, S₁ is 5-((5-aminopentyl) thioureidyl.

Preferably, where F is the fluorophore of fluorescein or one of its derivatives, S2 is selected from the group including: —CO(CH₂)₃CO—, —CO(CH₂)₄CO— (adipate), —CO(CH₂)₅CO— and —CO(CH₂)₅NHCO(CH₂)₅CO—. More preferably, where F is the fluorophore of fluorescein or one of its derivatives, S₂ is —CO(CH₂)₄CO— (adipate).

Preferably, where F is the fluorophore of fluorescein or one of its derivatives, the structure includes the substructure:

where m and n are independently 3 to 5 and

is other than H.

Preferably, where F is the fluorophore of BODIPY or one of its derivatives, S₁ is a C₃₋₅-alkionyldiamine. More preferably, where F is the fluorophore of BODIPY or one of its derivatives, S₁ is propionyl ethyldiamine.

Preferably, where F is the fluorophore of BODIPY or one of its derivatives, S₂ is selected from the group consiting of: —CO(CH₂)₃CO—, —CO(CH₂)₄CO— (adipate) and —CO(CH₂)₅CO—. More preferably, where F is the fluorophore of BODIPY or one of its derivatives, S₂ is —CO(CH₂)₄CO— (adipate).

Preferably, where F is the fluorophore of BODIPY or one of its derivatives the structure includes the substructure:

where p, q and r are independently 3 to 5 and

is other than H. More preferably, the sum of p, q and r is 8. Most preferably, p is 2, q is 2 and r is 4.

Preferably L is a lipid selected from the group consisting of diacyl- and dialkyl-glycerolipids, including glycerophospholipids. More preferably L is selected from the group consisting of: diacylglycerolipids, phosphatidate, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine, phosphatidyl inositol, phosphatidyl glycerol, and diphosphatidyl glycerol derived from one or more of trans-3-hexadecenoic acid, cis-5-hexadecenoic acid, cis-7-hexadecenoic acid, cis-9-hexadecenoic acid, cis-6-octadecenoic acid, cis-9-octadecenoic acid, trans-9-octadecenoic acid, trans-11-octadecenoic acid, cis-11-octadecenoic acid, cis-11-eicosenoic acid or cis-13-docsenoic acid. More preferably the lipid is derived from one or more cis-desaturated fatty acids. Most preferably L is selected from the group consisting of: 1,2-O-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE), 1,2-O-distearyl-sn-glycero-3-phosphatidylethanolamine (DSPE) and rac-1,2-dioleoylglycerol (DOG).

In a eleventh aspect the invention provides a method of marking cells including the step of contacting a suspension of cells with a cell marker of the tenth aspect of the invention.

In a twelth aspect the invention provides a cell incorporating a cell marker of the tenth aspect of the invention.

In a thirteenth aspect the invention provides a cell produced by the method of the eleventh aspect of the invention.

In the context of the description and claims: “BODIPY” means the compound assigned the Chemical Abstracts Service (CAS) Registry number 138026-71-8 and the CA index name: Boron, difluoro[2-[(2H-pyrrol-2-ylidene-KN)methyl]-1H-pyrrolato-pyrrolato-KN], (T-4)- (9CI); “fluorescein” means the chemical structure assigned the Chemical Abstracts Service (CAS) Registry number 518-47-8 and the CA index name: Spiro[isobenzofuran-1(3H),9′-[9H]xanthen]-3-one, 3′, 6′-dihydroxy-, sodium salt (1:2); “fluorophore” means the substructure or portion of a fluorescent molecule to which the fluorescent properties of the molecule are attributed, and “or one of its derivatives” means a chemical modification of the chemical structure to provide a fluorophore with substantially equivalent physico-chemical properties, but modified spectral characteristics.

DETAILED DESCRIPTION

The synthetic molecule constructs of the invention spontaneously and stably incorporate into a lipid bi-layer, such as a membrane, when a solution of the molecule is contacted with the lipid bi-layer. Whilst not wishing to be bound by theory it is believed that the insertion into the membrane of the lipid tails of the lipid (L) is thermodynamically favoured. Subsequent disassociation of the synthetic molecule construct from the lipid membrane is believed to be thermodynamically unfavoured. Surprisingly, the synthetic molecule constructs identified herein have also been found to be water soluble.

The synthetic molecule constructs of the invention are used to transform cells resulting in qualitative and/or quantitative changes in the surface antigens expressed. It will be recognised that the transformation of cells in accordance with the invention is distinguished from transformation of cells by genetic engineering. The invention provides for phenotypic transformation of cells without genetic transformation.

In the context of this description the term “transformation” in reference to cells is used to refer to the insertion or incorporation into the cell membrane of exogenously prepared synthetic molecule constructs thereby effecting qualitative and quantitative changes in the cell surface antigens expressed by the cell.

The synthetic molecule constructs of the invention comprise an antigen (including fluorophores) (F) linked to a lipid portion (or moiety) (L) via a spacer (S₁-S₂). The synthetic molecule constructs can be prepared by the condensation of a primary aminoalkyl, secondary aliphatic aminoalkyl or primary aromatic amine derivative of the antigen with an activated lipid. Methods of preparing neoglycoconjugates have been reviewed (Bovin (2002)).

A desired phenotypic transformation may be achieved using the synthetic molecule constructs of the invention in a one-step method or a two-step method. In the one step method the water soluble synthetic molecule construct (F—S₁-S₂-L) comprises the surface antigen as F.

In the two-step method the synthetic molecule construct (F—S₁-S₂-L) comprises an antigen (F) that serves as a functional group to which a surface antigen can be linked following insertion of the synthetic molecule construct into the membrane. The functional group can be a group such as a lectin, avidin or biotin. When used in the two step method the synthetic molecule construct is acting as a synthetic membrane anchor.

In accordance with the invention the primary aminoalkyl, secondary aliphatic aminoalkyl or primary aromatic amine and the activator of the lipid are selected to provide a synthetic molecule construct that is water soluble and will spontaneously and stably incorporate into a lipid bi-layer when a solution of the synthetic molecule construct is contacted with the lipid bi-layer.

In the context of this description the phrase “water soluble” means a stable, single phase system is formed when the synthetic molecule construct is contacted with water or saline (such as PBS) in the absence of organic solvents or detergents, and the term “solution” has a corresponding meaning.

In the context of this description the phrase “stably incorporate” means that the synthetic molecule constructs incorporate into the lipid bi-layer or membrane with minimal subsequent exchange between the lipid bi-layer or membrane and the external aqueous environment of the lipid bi-layer or membrane.

The selection of the primary aminoalkyl, secondary aliphatic aminoalkyl or primary aromatic amine and the activator depends on the physico-chemical properties of the antigen (F) to be linked to the lipid (L).

It will be understood by those skilled in the art that for a non-specific interaction, such as the interaction between a diacyl- or dialkyl-glycerolipid and a membrane, structural and stereo-isomers of naturally occurring lipids can be functionally equivalent. For example, it is contemplated by the inventors that diacylglycerol 2-phosphate could be substituted for phosphatidate (diacylglycerol 3-phosphate). Furthermore, it is contemplated by the inventors that the absolute configuration of phosphatidate can be either R or S.

The inventors have determined that to prepare synthetic molecule constructs (F—S₁-S₂-L) of the invention where the antigen (F) is a carbohydrate (or other antigen) with similar physico-chemical properties to the oligosaccharide of the A-, B- or H-antigens of the ABO blood groups and the lipid moiety (L) is a glycerophospholipid, S₁ is selected from —O (CH₂)_(n)NH— and S₂ is selected from —CO(CH₂)_(n)CO— or —CO(CH₂)_(m)NHCO (CH₂) _(n)CO— (where n and m are independently 2 to 5).

It will be understood by one skilled in the art that once the structure of the spacer (S₁-S₂) has been determined for a given class of antigens, e.g. carbohydrates, the same structure of the spacer can be adopted to prepare synthetic molecule constructs of other classes of antigen, e.g. fluorophores, with similar physico-chemical properties.

The structure of the spacer for synthetic molecule constructs (F—S₁-S₂-L) of the invention where F is a glycotope of the A-, B- and H-antigens of the ABO blood groups, may be the structure of the spacer selected to prepare synthetic molecule constructs comprising other glycotopes with physico-chemical properties similar to the glycotopes of the A-, B- and H-antigens of the ABO blood groups.

In principle the glycotope of a broad range of blood group related glycolipids or glycoproteins could be the antigen (F) of the synthetic molecule construct F—S₁-S₂-L where S₁-S₂-L is identical or equivalent to the corresponding portion of the synthetic molecule constructs designated A_(tri)-sp-Ad-DOPE (I), A_(tri)- spsp₁-Ad-DOPE (II), A_(tri)-sp-Ad-DSPE (III), B_(tri)-sp-Ad-DOPE (VI), H_(tri)-sp-Ad-DOPE (VII), H_(di) -sp-Ad-DOPE (VIII), Galβ-sp-Ad-DOPE (IX), Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAc-sp-Ad-DOPE (XII), and Fucαl-2Galβ1-3(Fucαl-4)GlcNAc-sp-Ad-DOPE (XIII).

TABLE 1 The structures (where G1 is D-Gal, Gc is D-Glc, GcN is D-GlcNAc, M is D-Man, F is L-Fuc and NA is NeuAc) of known blood group-related glycolipids and glycoproteins. Glycolipids A-12-2

A-14-2

A-16-2

A-6-1

A-6-2

A-7-1 (ALe^(b))

A-7-2 (ALe^(y))

A-7-4

A-8-2

A-9-3

B-12-2

B-14-2

B-16-2

B-6-1

B-6-2

B-8-2

BLe^(b)-7

BLe^(y)-7

Cad erythrocyte

Cad hepato- carcinoma antigen

Disialoyl Le^(a)-7

Erythrocyte FI antigen

Forssman antigen GalNAcα1→3GalNAcβ1→3Galα1→4Galβ1→4Glcβ1→1Cer Globoside/P GalNAcβ1→3Galα1→4Galβ1→4Glcβ1→1Cer Globotriaosylce ramide/P^(K) Galα1→4Galβ1→4Glcβ1→Cer H-5-1

H-5-2

H-6-4

H-8-3

Hematoside/G_(M3) NeuAcα2→3Galβ1→4Glcβ1→1Cer I antigen

i antigen/lacto-N-nor- Galβ1→4GlcNAclβ1→3Galβ1→4GlcNAcβ1→3Galβ1→4Glcβ1→1Cer hexaosylceramide I-active polyglycosylcer amide

Lactosylceramide Galβl→4Glcβ1→Cer Lactotriaosylceramide GlcNAcβ1→3Galβ1→4Glcβ1→Cer Le^(a)-11

Le^(a)-5

Le^(b)-6

Le^(b)-8

Le^(c)- Galβ1→3GlcNAcβ1→3Galβ1→4Glcβ1→1Cer 4/Lactotetraosylceramide Le^(c)-9 (fucosylated backbone)

Le^(c)-9 (fucosylated branch)

Le^(x)-11

Le^(x)-5

Le^(x)-7

Le^(x)-8

Le^(y)-6

LKE/′GL 7/SSEA-4 NeuAcα2→3Galβ1→3GalNAcβ→3Galα1→4Galβ1→4Glcβ1→1Cer P₁ Galα1→4Galβ1→4GlcNAcβ1→3Galβ1→4Glcβ1→1Cer Paragloboside/ Galβ1→4GlcNAcβ1→3Galβ1→4Glcβ1→1Cer neolactotetraosylceramide P-like GalNAcβ1→3Ga1β1→4GlcNAcβ1→3Galβ1→4Glcβ1→1Cer Sialoyl NeuAcα2→3Galβ1→4GlcNAcβ1→3Galβ1→4Glcβ1→1Cer paragloboside/ sialoylneolactotetraosylceramide Sialyl Le^(a)- 6/gastrointestinal cancer antigen (GICA or Ca 19-9)

Sialyl Le^(x)

Sialyl-nor-hexaosylceramide/ NeuAcα2→3Galβ1→4GlcNAcLβ1→3Galβ1→4GlcNAcβ1→3Galβ1→4Glcβ1→Cer sialoyl-lacto-N-nor- hexaosylceramide VIM-2

O-linked Glycoproteins Cad oligosaccharide

Disialotetrasac charide

Disialoyl group oligosaccharide

GlcNAc oligosaccharide

H-active trisaccharide

Monosialotrisac charide

Mucin oligosaccharide/ A-active glycoprotein

Ovarian cyst A- active glycoprotein-18

Ovarian cyst A- active glycoprotein-6a

Ovarian cyst A- active glycoprotein-6b

Ovarian cyst Le^(a)-active glycoprotein-10

Ovarian cyst Le^(a)-active glycoprotein-7

Sialylated H-active tetrasaccharide

N-linked Glycoproteins Adult lactosaminoglycan

Complex type/Alkali-stable chain

Difucosyl adult erythrocyte antigen

Difucosyl foetal erythrocyte antigen

Disialyl foetal erythrocyte antigen

Foetal lactosaminoglycan

High-mannose type

Hybrid type

Monofucosyl-disialyl foetal erythrocyte antigen (disialyl group on branch)

Monofucosyl- monosialoyl adult erythrocyte antigen

Monofucosyl- monosialoyl adult erythrocyte antigen

Monofucosyl-monosialyl foetal erythrocyte antigen (fucosylated backbone)

Monofucosyl-monosialyl foetal erythrocyte antigen (fucosylated branch)

Tamm-Horsfall glycoprotein

Trisialoyl foetal erythrocyte antigen (disialoyl group on branch)

In general, for almost all examples of A-antigens the terminal A sugar N-acetylgalactoseamine (GalNAc) can be replaced with the B sugar galactose

(Gal). Additionally, the lack of either the A or B determinant creates the equivalent H determinant.

It will be understood by those skilled in the art that the synthetic molecule constructs (F—S₁-S₂-L) of the invention where F is an oligosaccharide may be used as “synthetic glycolipids” and substituted for glycolipids obtained from biological (botanical or zoological) sources.

In the context of this description of the invention the term “glycolipid” means a lipid containing carbohydrate of amphipathic character including: glycosylated glycerolipids, such as glycosylated phosphoglycerides and glycosylglycerides; glycosylated sphingolipids (neutral glycolipids) such as glycosylceramides or cerebrosides; and gangliosides (acidic glycolipids).

In the context of this description of the invention the phrase “glycolipid-linked antigen” means a lipid containing carbohydrate in which an antigen (e.g. a protein) is linked to the glycolipid via the carbohydrate portion of the molecule. Examples of glycolipid-linked antigens include GPI-linked proteins.

It will be understood by those skilled in the art that a glycolipid is itself an antigen. The term and phrase “glycolipid” and “glycolipid-linked antigen” are used to distinguish between naturally occurring molecules where the antigen is the glycolipid and naturally occurring molecules where the antigen is linked to the glycolipid via the carbohydrate portion of the glycolipid. By analogy the synthetic molecule constructs of the invention could be described as both “synthetic glycolipids” and “synthetic membrane anchors” to the extent that the antigen may be the synthetic glycolipid per se or attached to the synthetic glycolipid.

It will be understood by those skilled in the art that the carbohydrate portion of a glycolipid may be modified and linked to other antigens by the methods described in the specification accompanying the international application no. PCT/NZ2003/00059 (publ. no. WO 03/087346).

In the context of this description of the invention the term “glycotope” is used to refer to the antigenic determinant located on the carbohydrate portion of a glycolipid. The classification of glycolipid antigens in blood group serology is based on the structure of the carbohydrate portion of the glycolipid.

In blood group serology it is known that the terminal sugars of the glycotopes of A-antigens are GalNAcα1-3(Fucα1-2)Galβ, and the terminal sugars of the glycotopes of the B-antigens are Galα1-3(Fucα1-2)Galβ. Incorporation into the membrane of RBCs of water soluble synthetic molecule constructs of the invention where F is GalNAcα1-3(Fucα1-2)Galβ or Galα1-3(Fucα1-2)Galα provides RBCs that are serologically equivalent to A-antigen or B-antigen expressing RBCs, respectively.

The terminal three sugars of the carbohydrate portion of the naturally occurring A- or B-antigen are the determinant of the A and B blood groupings.

The terminal four or five sugars of the carbohydrate portion of the naturally occurring A-antigen are the determinant of the A blood sub-groupings A type 1, A type 2, etc. Accordingly, the RBCs incorporating the synthetic molecule constructs of the invention can be used to characterise and discriminate between blood typing reagents (antibodies) of differing specificity.

Water soluble synthetic molecule constructs of the invention that exclude a carbohydrate portion are contemplated by the inventors. Antigens other than carbohydrates or oligosaccharides, but with similar physico-chemical properties, may be substituted for F in the “synthetic glycolipids” described.

Synthetic molecule constructs of the invention that comprise an antigen (F) with differing physico-chemical properties to those of carbohydrates or oligosaccharides are also contemplated by the inventors. Water soluble synthetic molecule constructs comprising these antigens may be prepared by selecting different spacers.

The advantages provided by the synthetic molecule constructs of this invention will accrue when used in the practice of the inventions described in the specifications accompanying international application nos. PCT/NZ02/00212 (publ. no. WO 03/034074) and PCT/NZ03/00059 (publ. no. WO 03/087346). The disclosures of the specifications accompanying these applications are incorporated herein by reference.

The synthetic molecule constructs overcome many of the limitations of using natural glycolipids in the practice of these inventions. A particular advantage of the synthetic molecule constructs is their superior performance and ability to be used in the transformation of cells at reduced temperatures, e.g. 4° C.

As described herein not all structures of the spacer (S₁-S₂) will provide a synthetic molecule construct (F—S₁-S₂-L) that is water soluble and spontaneously and stably incorporate into a lipid bi-layer such as a cell membrane. The synthetic molecule constructs designated A_(tri)-sp-lipid (IV) and A_(tri)-PAA-DOPE (V) were determined not to be water soluble and/or unable to spontaneously and stably incorporate into a lipid bilayer such as a cell membrane.

where x and y are in the range 0.05 to 0.2

The invention will now be illustrated by reference to the following non-limiting examples and figures of the accompanying drawings in which:

FIG. 1 shows Diamed results of Cellstab™ stored cells transformed by natural A glycolipid transformation solution at (L to R) 10 mg/mL, 5 mg/mL, 2 mg/mL, 2 mg/mL* and 1 mg/mL. Antisera used are Albaclone (top) and Bioclone (bottom). (*—transformation solution (containing glycolipids) was not washed out after the incubation, it was left in over night and washed out the next day (day 2).)

FIG. 2 shows Diamed results of CellstabTM stored cells transformed by natural B glycolipid transformation solution at (L to R) 10 mg/mL, 5 mg/mL, 2 mg/mL, 2 mg/mL* and 1 mg/mL. Antisera used are Albaclone (top) and Bioclone (bottom). (*—transformation solution (containing glycolipids) was not washed out after the incubation, it was left in over night and washed out the next day (day 2)).

FIG. 3 shows FACS analysis following in vitro transformation of human Le(a-b-) red cells with natural Le^(b)-6 glycolipid over time at three transformation temperatures, 37° C. (top), 22° C. (middle) and 4° C. (bottom).

FIG. 4 shows Diamed results of cells transformed at 4° C. by A_(tri)-sp-Ad-DOPE (I) transformation solution at (L to R): washed 0.08 mg/mL; unwashed 0.08 mg/mL; washed 0.05 mg/mL; unwashed 0.05 mg/mL; washed 0.03 mg/mL; and unwashed 0.03 mg/mL. The antisera used was Bioclone anti-A.

FIG. 5 shows cells that were no longer washed prior to testing. Diamed results of cells transformed at 4° C. by A_(tri)-sp-Ad-DOPE (I) transformation solution at (L to R): 0.08 mg/mL, 0.05 mg/mL and 0.03 mg/mL. The antisera used was Bioclone anti-A.

FIG. 6 shows in the left column Diamed results of cells transformed at 4° C. by B_(tri)-sp-Ad-DOPE (VI) transformation solution at (L to R): washed 0.6 mg/mL;

unwashed 0.6 mg/mL; washed 0.3 mg/mL; unwashed 0.3 mg/mL; washed 0.15 mg/mL; and unwashed 0.15 mg/mL; and in the right column Diamed results of cells transformed at 4° C. by B_(tri)-sp-Ad-DOPE (VI) transformation solution at (L to R): washed 0.08 mg/mL; unwashed 0.08 mg/mL; washed 0.05 mg/mL; unwashed 0.05 mg/mL; washed 0.03 mg/mL; and unwashed 0.03 mg/mL. The antisera used was Bioclone anti-B.

FIG. 7 shows cells that were no longer washed prior to testing. Diamed results of cells transformed at 4° C. by B_(tri)-sp-Ad-DOPE (VI) transformation solution at (L to R): 0.6 mg/mL, 0.3 mg/mL and 0.15 mg/mL.

FIG. 8 shows Diamed results of cells transformed at 4° C. by parallel transformation with A_(tri)-sp-Ad-DOPE (I) and B_(tri)-sp-Ad-DOPE (VI). Wells 1 and 2(L to R) contain washed A 0.07 +B 0.3 mg/mL against anti-A and anti-B. Wells 3 and 4 contain unwashed A 0.07 +B 0.3 mg/mL against anti-A and anti-B.

FIG. 9 shows cells that were no longer washed prior to testing. Diamed results of cells transformed at 4° C. by parallel transformation with A_(tri)-sp-Ad-DOPE (I) and B_(tri)-sp-Ad-DOPE (VI). Wells 1 and 2 (L to R) contain unwashed A 0.07+B 0.3 mg/mL against anti-A and anti-B.

FIG. 10 shows Diamed results of cells transformed at 4° C. by parallel transformation with A_(tri)-sp-Ad-DOPE (I) and B_(tri)-sp-Ad-DOPE (VI). Wells 1 and 2 (L to R) contain washed A 0.07+B 0.2 mg/mL against anti-A and anti-B. Wells 3 and 4 contain unwashed A 0.07+B 0.2 mg/mL against anti-A and anti-B.

FIG. 11 shows cells that were no longer washed prior to testing. Diamed results of cells transformed at 4° C. by parallel transformation with A_(tri)-sp-Ad-DOPE (I) and B_(tri)-sp-Ad-DOPE (VI). Wells 1 and 2 (L to R) contain unwashed A 0.07+B 0.2 mg/mL against anti-A and anti-B.

FIG. 12 shows Diamed results of cells transformed at 4° C. by parallel transformation with A_(tri)-sp-Ad-DOPE (I) and B_(tri)-sp-Ad-DOPE (VI). Wells 1 and 2 (L to R) contain washed A 0.06 +B 0.3 mg/mL against anti-A and anti-B. Wells 3 and 4 contain unwashed A 0.06+B 0.3 mg/mL against anti-A and anti-B.

FIG. 13 shows cells that were no longer washed prior to testing. Diamed results of cells transformed at 4° C. by parallel transformation with A_(tri)-sp-Ad-DOPE (I) and B_(tri)-sp-Ad-DOPE (VI). Wells 1 and 2 (L to R) contain unwashed A 0.06+B 0.3 mg/mL against anti-A and anti-B.

FIG. 14 shows Diamed results of cells transformed at 4° C. by parallel transformation with A_(tri)-sp-Ad-DOPE (I) and B_(tri)-sp-Ad-DOPE (VI). Wells 1 and 2 (L to R) contain washed A 0.06 +B 0.2 mg/mL against anti-A and anti-B. Wells 3 and 4 contain unwashed A 0.06 +B 0.2 mg/mL against anti-A and anti-B.

FIG. 15 shows cells that were no longer washed prior to testing. Diamed results of cells transformed at 4° C. by parallel transformation with A_(tri)-sp-Ad-DOPE (I) and B_(tri)-sp-Ad-DOPE (VI). Wells 1 and 2 (L to R) contain unwashed A 0.06+B 0.2 mg/mL against anti-A and anti-B.

FIG. 16 shows Diamed results of cells transformed at 4° C. by parallel transformation with A_(tri)-sp-Ad-DOPE (I) and B_(tri)-sp-Ad-DOPE (VI). Wells 1 and 2 (L to R) contain washed A 0.05+B 0.3 mg/mL against anti-A and anti-B. Wells 3 and 4 contain unwashed A 0.05 +B 0.3 mg/mL against anti-A and anti-B.

FIG. 17 shows cells that were no longer washed prior to testing. Diamed results of cells transformed at 4° C. by parallel transformation with A_(tri)-sp-Ad-DOPE (I) and B_(tri)-sp-Ad-DOPE (VI). Wells 1 and 2 (L to R) contain unwashed A 0.05+B 0.3 mg/mL against anti-A and anti-B.

FIG. 18 shows Diamed results of cells transformed at 4° C. by parallel transformation with A_(tri)-sp-Ad-DOPE (I) and B_(tri)-sp-Ad-DOPE (VI). Wells 1 and 2 (L to R) contain washed A 0.05+B 0.2 mg/mL against anti-A and anti-B. Wells 3 and 4 contain unwashed A 0.05 +B 0.2 mg/mL against anti-A and anti-B.

FIG. 19 shows cells that were no longer washed prior to testing. Diamed results of cells transformed at 4° C. by parallel transformation with A_(tri)-sp-Ad-DOPE (I) and B_(tri)-sp-Ad-DOPE (VI). Wells 1 and 2 (L to R) contain unwashed A 0.05+B 0.2 mg/mL against anti-A and anti-B.

FIG. 20. Red blood cells following contact with a synthetic molecule construct (XVI) viewed with a fluorescence microscope at 470 nm under 250× magnification.

COMPARATIVE EXAMPLES

The Comparative Examples do not form part of the invention claimed. The Comparative Examples describe red blood cell (RBC) transformation with natural glycolipids.

Comparative Example 1 Preparation of Natural Glycolipids

Purification by HPLC

In the first stage, columns were packed with dry silica (15-25 μm) before each run. Relatively dirty samples could be used in HPLC because the silica could be discarded along with the theoretically high level of irreversibly bound contaminants.

Glycolipids were separated on silica gel with a mobile phase of increasing polarity. The program was a linear gradient beginning with 100% chloroform-methanol-water 80:20:1 (v/v) and ending with 100% chloroform-methanol-water 40:40:12 (v/v).

The HPLC equipment used was a Shimadzu system capable of pumping and mixing four separate solvents at programmed ratios. As chloroform, methanol and water evaporate at different rates, a program was developed whereby the solvent components were not mixed prior to entering the HPLC.

The Shimadzu HPLC mixes four different liquids by taking a “shot” from each of four bottles in turn. “Shots” of chloroform and water directly next to each other in the lines may cause miscibility problems. Methanol was sandwiched in between these two immiscible components. Additionally, the water was pre-mixed with methanol in a 1:1 ratio to further prevent problems with miscibility.

Comparative Example 2 Transformation of Red Blood Cell Transformation with Natural Glycolipids

Agglutination

Transformation of red blood cells was assessed by agglutination using the Diamed-ID Micro Typing System in addition to using conventional tube serology. Diamed ABO typing cards were not used. The cards used were NaCl, Enzyme test and cold agglutinin cards, which were not pre-loaded with any antisera or other reagents. This allowed the use of specific antisera with both methodologies.

A comparative trial was carried out between tube serology and the Diamed system to establish the performance of the two systems. Cells were transformed at 25° C. for 4 hours. Seraclone and Alba-clone anti-A sera were used to gauge equivalency. The results are shown in Table 3 below.

TABLE 2 Antisera used in comparison of tube serology with the Diamed system. Manufacturer Catalogue ref Lot Expiry Albaclone, SNBTS Anti-A. Z0010770 12.12.04 Seraclone, Biotest 801320100 1310401 12.04.03

TABLE 3 Agglutination results comparing tube serology with the Diamed system. A glycolipid (mg/mL) 10 5 2 1 0 Tube Albaclone 3+ 2+ 0 0 0 Seraclone 3+ 2+ 0 0 0 Diamed Albaclone 2+ 2+ 0 0 0 Seraclone 3+ 2+ 1+ w+ 0

In this experiment, the Diamed system proved to be more sensitive to the weaker reactions than tube serology with the Seraclone anti-A, but not with Albaclone. These reagents are formulated differently and are thus not expected to perform identically. However, the fact that the Seraclone anti-A tube serology combination did not detect positivity is probably due to operator interpretation. The weaker reactions are notoriously difficult to accurately score, and the difference between 1+and 0 can be difficult to discern in tubes.

Optimisation

The variables of glycolipid concentration, incubation temperature, incubation duration, diluent and storage solution were examined for their effect on cell health. Efficiency and stability of transformation was assessed by agglutination with the relevant antibody.

TABLE 4 Tube serology agglutination of natural glycolipid A transformed cells over different times and temperatures. A 10 5 2 1 0.1 0.01 0.001 0.0001 0 Seraclone 3+ 2+ 0  0  0 (37° C. for 1.5 hours) Seraclone 4+ 3+ 2+ 1+ w+ 0 0 0 0 (25° C. for 4 hours)

Glycolipid Concentration

Initial transformation experiments were carried out with a highly purified (HPLC) Le^(b) glycolipid sample and a less pure blood group A glycolipid sample. Transformation was performed at 37° C. for 1.5 hours

The A glycolipid sample contained other lipid impurities and thus comparatively less blood group A molecules by weight than the Le^(b) glycolipid sample of equivalent concentration (w/v). This seems to be borne out by the fact that higher concentrations of the A glycolipid than the Le^(b) glycolipid were required to produce equivalent agglutination scores (see Table 6).

The level of impurity in the A glycolipid sample may also have contributed to the lower stability over the 62 day period—the A-transformed cells ‘died’ at the highest concentration (having received the largest dose of impurity).

TABLE 5 Anti-A and anti-Le^(b) used in initial testing of natural glycolipid transformation. Manufacturer Catalogue ref Batch number Expiry Anti-A 801320100 1310401 12.04.03 Seraclone, Biotest Anti-Le^(b) 12801 CSL

TABLE 6 Stability of RBCs transformed with natural A and Le^(b) glycolipid as assessed by tube serology agglutination over the period of 62 days. Glycolipid Le^(b) A (mg/mL) Day 1 Day 25 Day 62 Day 1 Day 25 Day 62 10 4+ 2-3+  3+ 2+ ? 5 4+ 2-3+  2+ 2+ w+ 2 3+ 1-2+ 0 1+ 0 1 4+  2+ 0 1+ 0 0.1 3+ 2+ 0 0 0.01 2+ 2+ 0 0 0.001 2+ 2+ 0 0 0.0001 2+ 0  0 0 0 0  0  0 0 0  0

The above cells were also rated for haemolysis and these results are shown in Table 7 below.

TABLE 7 Haemolysis as assessed visually. Glycolipid Haemolysis concentration Le^(b) A (mg/mL) Day 1 Day 25 Day 62 Day 1 Day 25 Day 62 10 h 0 h h h dead 5 hh 0 hhh w 0 hh 2 w 0 hhh w 0 hhhhh 1 w 0 hhh h 0 hhhh 0.1 h hhh 0.01 hh 0.001 h 0.0001 h Control h 0 h h h Day 1—in the supernatant of the first wash after transformation; Days 25 and 62—in the cell preservative solution before the cells are resuspended after storage. Scoring scale is analogous to the 4+ to 0 agglutination scale: hhhh—severely haemolysed, hhh—very haemolysed, hh—moderately haemolysed, h—mildly haemolysed, w—faintly haemolysed and 0—no haemolysis seen.

These results show that cell haemolysis can be shown to be associated with transformation with high concentrations of glycolipid. It is unclear whether the mechanism underlying this is disruption of the plasma membrane by large amounts of glycolipid being inserted, the rate of that insertion, or is possibly due to the quantity of associated impurity. However, the results for Le^(b) at day 62 seem to support the first explanation.

The Le^(b) sample was highly purified - before being dissolved, it was a powder of pure white colour, and thus it is unlikely that the haemolysis was due to the deleterious effect of impurities. It is clear to see that at 62 days, the amount of haemolysis occurring diminishes in line with the decrease in the glycolipid concentration.

Incubation Temperature

Experiments were carried out to investigate other possible mechanisms for the reduction of haemolysis of RBCs during the insertion step. Previous experiments had shown that haemolysis was worse at higher glycolipid concentrations than at lower concentrations, and it is thought that haemolysis may also be related to the rate of glycolipid insertion. Since temperature is believed to affect the rate of insertion, experiments were conducted comparing transformation at 37° C. with transformation at room temperature (RT; 25° C.)

Since the rate was expected to slow down as temperature decreased, the incubation period for the RT experiment was 4 hrs. Haemolysis was assessed visually and scored following insertion. Serology tests were also performed on the cells. The results are shown in Table 8.

TABLE 8 The effect of incubation temperature on haemolysis and agglutination during insertion of glycolipids into RBC membranes. Haemolysis was scored visually at each of the three washes Haemolysis RT 37° C. Glycolipid wash wash wash wash wash wash Serology (mg/mL) 1 2 3 1 2 3 RT 37° C. 10 w 0 0 hh w 0 2+ 2+ 1 w 0 0 hh h vw 1+ w+

Incubation Duration

Incubation at 37° C. was carried out for 1 and 2 hours and its effect on cell health and transformation assessed by agglutination with the relevant antibody.

TABLE 9 Antisera used in the duration of incubation trial. Manufacturer Catalogue ref Batch number Expiry date Albaclone, SNBTS Anti-A. Z0010770 12.12.04 Bioclone, OCD Anti-A, DEV01102 — experimental reagent Albaclone, SNBTS Anti-B Z0110670 01.07.05 Bioclone, OCD Anti-B, DEV01103 — experimental reagent

TABLE 10 Effect of incubation time on agglutination of cells transformed with natural glycolipids. Concentration Albaclone BioClone Glycolipid (mg/mL) 1 hour 2 hours 1 hour 2 hours A 10 4+ 4+ 4+ 4+ 5 4+ 4+ 4+ 2+ 2 4+ 3+ 3+ 2+ 1 3+ 2+ 2+ 2+ 0.5 2+ 2+ 1+ w+ B 10 3+ 2+ 4+ 1+ 5 3+ 2+ 3+ 2+ 2 2+ 2+ 2+ 1+ 1 1+ w+ 1+ w+ 0.5 1+ w+ w+ w+

These results indicate that increasing the duration of incubation during natural glycolipid insertion does not enhance agglutination. In fact, the agglutination scores are reduced after the two hour incubation. This may be due to the destabilisation of the membrane or exchange of the glycolipids back into solution.

Diluent

Experiments were also carried out to determine if changing the glycolipid diluent solution could reduce haemolysis. Working strength PBS was compared with 2×PBS and 2% Bovine Serum Albumin (BSA) in working strength PBS. Cells were incubated at 37° C. for 1.5 hours. The results are shown in Table 11.

TABLE 11 Study on the effect on haemolysis of changing the glycolipid diluent solutions during insertion of glycolipids into RBC membranes. Glycolipid concentration Glycolipid Diluent Solution (mg/mL) PBS 2 × PBS 2% BSA in PBS 40 Hhh hhh hhh 30 Hhh hhh hhh 20 Hhh hhh hhh 10 Hhh hhh hhh 0 0 0 0

Stability

Once A and B blood group glycolipids had been HPLC purified to an acceptable level, an experiment to find the appropriate concentrations for stability trials was carried out.

TABLE 12 Early stability trial of cells transformed with natural A glycolipid. A Expt Day 10 5 2 1 0.1 0.01 0.001 0.0001 0 1 7 4+ 3-4+  1+ 0 0 0 0 0 0 2 43 3+ w+ 0 0 0 0 0 0 0 3 50 1+ 0  0 0 4 60 3+ 1+ 0 5 67 w+ vw vw 6 74 2+ 0  0 7 81 2+ 1+ 0

TABLE 13 Antisera used in stability trials (Table 14 and Table 15). Batch Expiry Manufacturer Catalogue ref number date Albaclone, SNBTS Anti-A. Z0010770 12.12.04 Bioclone, OCD Anti-A, experimental reagent DEV01102 — Albaclone, SNBTS Anti-B Z0110670 01.07.05 Bioclone, OCD Anti-B, experimental reagent DEV01103 —

TABLE 14 Tube serology of O RBCs transformed with A glycolipid in order to establish appropriate concentrations for stability trials. Transformation at 25° C. for 4 hours. A glycolipid (mg/mL) Anti-A Expt 10 5 2 1 0.5 0.1 0.01 0.001 0 Alba 1 3+ 2+ 1+ 0  0 0 0 0 2 4+ 4+ 3+ 2+ w+ Bio 1 3+ 2+ 1+ 0  0 0 0 0 2 4+ 4+ 3+ 2+ w+

TABLE 15 Tube serology of O RBCS transformed with B glycolipid in order to establish appropriate concentrations for stability trials. B glycolipid (mg/mL) Anti-B Expt 10 5 2 1 0.5 0.1 0.01 0.001 0 Alba 1 2+ 1+ w+ 0 0 0 0 0 2 1+ 1+ w+ 0 w+ Bio 1 3+ 2+ w+ 0 0 0 0 0 2 1+ 1+ w+ 0 w+

Two sets of cells were transformed with different concentrations of natural A glycolipid. Transformation was performed at 25° C. One set of cells was tested long term, and one set of cells was tested weekly for agglutination. The agglutination results from tube serology and Diamed are shown in Table 16 below. All cells were stored in Cellstab™ in bottles with flat bases. The cells showed minimal to no haemolysis at any time.

TABLE 16 Agglutination results for cells transformed with different concentrations of natural A glycolipid. Results were obtained using Albaclone anti-A * - Albaclone, while all others used Seraclone anti-A. A glycolipid (mg/mL) 10 5 2 1 0.1 control Long term testing Day 1 Tube 4+ 3+ 2+ 1+ +w 0 Diamed 3+ 3+ +w 0 0 0 Day 17 Tube 3+ 2+ 0 0 0 Diamed 3+ 2+ 1+ 0 0 Weekly testing Day 1 Tube 3+ 2+ 0 0 Diamed 3+ 0 0 0 Day 8 Tube 1+ 0 0 0 Diamed 3+ 0 0 0 Day 15 Tube 1+ 0 0 0 Diamed 3+ 2+ 0 0 Day 22 Tube 3+ 0 0 0 Diamed 3+ 0 0 0 Day 29 Tube *+w *0 *0 *0 Diamed *3− *0 *0 *0 Day 36 Tube * * * *0 Diamed *3− *0 *0 *0 Day 43 Tube * * * *0 Diamed * * * *0

Storage Solution

Comparison of the two cell storage solutions, Celpresol™ (CSL) and Cellstab™ (Diamed) was carried out to test their relative abilities to support modified RBCs.

The stability of RBCs transformed with blood group A and B antigen solutions of varying concentrations when stored in two different cell preservative solutions—Cellstab™ and Alsevers™—was trialed.

A and B antisera from two different sources were used in serology testing.

All cells were tested using the standard tube serology platform up to 42 days, at which time the cell agglutination reactions had become too difficult to score manually (see Table 17 for A results and Table 18 for B results).

Diamed gel-card testing was carried out to day 56 for the Alsevers stored cells, and discontinued at day 63 due to fungal contamination (although still returning positive scores). The Cellstab™ stored cells continued to be tested up to day 70, and were still viable at this point (see FIG. 1 for A results and FIG. 2 for B results).

The reagents used in the stability trial are shown in Table 13.

TABLE 17 Tube serology results of stability trial of cells transformed with varying concentrations of A glycolipid and stored in either Cellstab ™ or Alsevers ™. Albaclone Anti-A Bioclone Anti-A Cell (SNBTS) Transformation (OCD - Developmental storage Solution (mg/mL) reagent) Day solution 10 5 2 2* 1 10 5 2 2* 1 2 Alsevers 4+ 3+ 2+ 1+ w+ 3+ 3+ 1+ 1+ 0 Cellstab ™ 4+ 4+ 3+ 1+ 1+ 3+ 3+ 2+ 1+ 0 8 Alsevers 4+ 4+ 2+ 1+ 1+ 2+ 2+ 1+ 1+ 0 Cellstab ™ 4+ 4+ 3+ 2+ 1+ 3+ 3+ 2+ w+ 0 14 Alsevers 4+ 3+ 2+ 2+ w+ 2+ 1+ w+ vw 0 Cellstab ™ 4+ 3+ 3+ 2+ w+ 3+ 2+ w+ vw 0 21 Alsevers 3+ 2+ 2+ 2+ 1+ 2+ 2+ 2+ 1+ 0 Cellstab ™ 3+ 3+ 2+ + ^(‡) 2+ ^(‡) ^(‡) ^(‡) 0 28 Alsevers 2+ 2+ 1+ 1+ 0  2+ 2+ 1+ 1+ 0 Cellstab ™  2+^(‡)  2+^(‡) ^(‡) ^(‡) 0  1+ w+ 0  0  0 36 Alsevers 3+ 2+ 2+ 2+ 1+ 3+ 3+ 2+ 1+  1+ Cellstab ™  3+^(‡)  2+^(‡) ^(‡) ^(‡) ^(‡)  3+^(‡) ^(‡) ^(‡) ^(‡) ^(‡) 42 Alsevers 3+ 3+ 1+ w+ 0  2+ 2+ 2+ 1+  1+ Cellstab ™  4+^(‡)  4+^(‡) ^(‡) ^(‡) ^(‡) ^(‡) ^(‡) ^(‡) ^(‡) 0 *transformation solution (containing glycolipids) was not washed out after the incubation, it was left in over night and washed out the next day. ^(‡)positive cell button, but cells fall off as negative (score assignment impossible).

FACS Analysis of Glycolipid Insertion

Transformation of human Le(a-b-) red cells with natural Le^(b)-6 glycolipid over time at three transformation temperatures (37° C., 22° C. and 4° C.) was performed (FIG. 3). Natural Le^(b)-6 glycolipid was dissolved in plasma and used to transform RBCs at a final concentration of 2 mg/mL and a final suspension of 10%.

TABLE 18 Tube serology results of stability trial of cells transformed with varying concentrations of B glycolipid and stored in either Cellstab ™ or Alsevers ™. Albaclone Anti-B Bioclone Anti-B Cell (SNBTS) Transformation (OCD - Developmental storage Solution (mg/mL) reagent) Day solution 10 5 2 2* 1 10 5 2 2* 1 2 Alsevers 3+ 3+ 1+ 1+  1+ 2+ 1+ 1+ 1+ 0 Cellstab ™ 3+ 3+ 2+ 2+  1+ 2+ 2+ 2+ 1+ w+ 8 Alsevers 1+ 1+ w+ 0  0 0  0  0  0  0 Cellstab ™ 2+ 1+ w+ 0 1+ 1+ w+ 0  0 14 Alsevers 2+ 2+ 0  w+ 0 0  1+ 1+ 2+ 0 Cellstab ™ 1+ w+ 0  0  0 2+ 2+ w+ 1+  1+ 21 Alsevers ^(‡) ^(‡) ^(‡) ^(‡) ^(‡) 1  1  ^(‡) ^(‡) ^(‡) Cellstab ™ ^(‡) ^(‡) ^(‡) ^(‡) ^(‡) + + + ^(‡) ^(‡) 28 Alsevers 2+ 1+ w+ 0  0 2+ 1+ 2+ 0  0 Cellstab ™ ^(‡) ^(‡) ^(‡) 0  0 ^(‡) 0  ^(‡) ^(‡) 0 36 Alsevers 2+ 2+ 2+ 1+  1+ 2+ 2+ 2+ 1+  1+ Cellstab ™ ^(‡) ^(‡) ^(‡) ^(‡) ^(‡) ^(‡) ^(‡) ^(‡) ^(‡) ^(‡) 42 Alsevers 2+ 2+ 2+ 2+ w+ 2+ 2+ 1+ w+ w+ Cellstab ™ ^(‡) ^(‡) ^(‡) ^(‡) ^(‡) ^(‡) ^(‡) ^(‡) ^(‡) ^(‡) *transformation solution (containing glycolipids) was not washed out after the incubation, it was left in over night and washed out the next day. ^(‡) positive cell button, but cells fall off as negative (score assignment impossible).

Reactivity was determined by FACS analysis using a Gamma anti-Leb. (The serological detection level is around 10² molecules. The insertion of natural glycolipids at 4° C. for 8 hours was not detectable by agglutination with antibodies.) Projection of the rate of insertion curve from FACS analysis did not indicate that the rate of insertion at 4° C. would have reached agglutination detection levels within 24 hours.

Low Incubation Temperature

Transformation of RBCs with natural A or B glycolipid was perfomed at 37° C. for 1 hour and 2° C. for varying intervals. Cells were agglutinated with Bioclone anti-A or Bioclone anti-B. The results are provided in Tables 19 and 20.

TABLE 19 Diamed results of comparison of natural A glycolipid transformation at 37° C. for 1 hour and 2° C. for varying intervals. Time Nat A (mg/mL) Temp (hours) 10 5 2 1 0 37° C. 1 3+ 3+ 2-3+  2+ 0  2° C. 1 0  0  0 0 0 4 0  0  0 0 0 8 1-2+ 0  0 0 0 24 2-3+ 2+ 1-2+ 0 0 48 3+ 2-3+ 2-3+ 0 0 72 3-4+ 3+  2+ 0 0

TABLE 20 Diamed results of comparison of natural B glycolipid transformation at 37° C. for 1 hour and 2° C. for varying intervals. Time Nat B (mg/mL) Temp (hours) 10 5 2 1 0 37° C. 1 3+ 2-3+  2+ 0 0  2° C. 1 0  0 0 0 0 4 0  0 0 0 0 8 0  0 0 0 0 24 1+ 0 0 0 0 48 2+ 1-2+ 0 0 0 72 2+  1+ 0 0 0

The rate of transformation is slow for both natural A glycolipid and natural B glycolipid as demonstrated by the negative agglutination scores after 1 hour at 2° C. Considerable insertion at 37° C. for this time interval has been demonstrated. Natural A glycolipid insertion at 2° C. required 48 hours to reach the same level of insertion obtainable by transformation at 37° C. After this time further insertion was not observed. Likewise, natural B glycolipid insertion at 2° C. was not as rapid as transformation at 37° C. The agglutination scores did not improve upon continued incubation and thus seemed to have reached maximal insertion at this time point for these concentrations.

Examples

The Examples describe red blood cell transformation with the synthetic molecule constructs of the invention. In the context of these examples the term “synthetic glycolipids” is used to refer to these constructs.

Example 1 Preparation of Synthetic Glycolipids

Materials and General Methods

Acetone, benzene, chloroform, ethylacetate, methanol, o-xylene, toluene, and 2-propanol were from Khimed (Russian Federation). Acetonitrile was from Cryochrom (Russian Federation). DMSO, DMF, CF₃COOH, Et₃N, N,N′-dicyclohexylcarbodiimide and N-hydroxysuccinimide were from Merck (Germany). N-methylmorpholin (NMM), 2-maleimidopropionic acid and disuccimidilcarbonate were from Fluka. Iminodiacetic acid dimethyl ester hydrochloride was from Reakhim (Russian Federation). Molecular sieves (MS 3 Å and 4 Å), trimethylsilyl trifluoromethanesulfonate, and triphenylphosphine were from Aldrich (Germany). All hydrides, 1,8-diazabicyclo[5,4,0]undec-7-ene (DBU), and trichloroacetonitrile were from Merck (Germany).

Anhydrous tetrahydrofuran (THF) and diethyl ether (Et₂O) were obtained by distillation from lithium aluminium hydride (H₄AlLi). Dichloromethane for glycoside synthesis was dried by distillation from phosphorous pentoxide and calcium hydride and stored over molecular sieves MS 4 Å. Solid reagents were dried for 2 h in vacuo (0.1 mm Hg) at 20 to 40° C. Deacetylation was performed according to Zemplen in anhydrous methanol. The solution of the acetylated compound was treated with 2 M sodium methylate in methanol up to pH 9. When the reaction was completed, Na⁺ ions were removed with cation exchange resin Dowex 50X-400 (H⁺) (Acros, Belgium). The solution was concentrated in vacuo.

Column chromatography was carried out on Silica gel 60 (0.040-0.063 mm, Merck, Germany). Gel chromatography was performed on Sephadex LH-20 (Pharmacia, Sweden). Solvents were removed in vacuo at 30 to 40 ° C. Thin-layer chromatography was performed on Silica gel 60 (Merck, Germany) precoated plates. Spots were visualized by treating with 5% aqueous orthophosphoric acid and subsequent heating to 150° C. in the case of carbohydrates or by soaking in ninhydrin solution (3 g/1 in 30:1 (v/v) butanol-acetic acid) in the case of amines.

Optical rotation was measured on a Jasco DIP-360 digital polarimeter at 25° C. Mass spectra were recorded on a Vision-2000 (Thermo Bioanalysis, UK) MALDI-TOF mass spectrometer using dihydroxybenzoic acid as a matrix. ¹H NMR spectra were recorded on a Bruker WM spectrometer (500 MHz) at 25° C. Chemical shifts (δ, ppm) were recorded relative to D₂O (δ=4.750), CDCl₃ (δ=7.270), and CD₃OD (δ=3.500) as internal standards. The values of coupling constants (Hz) are provided. The signals in the ¹H NMR spectra were assigned by suppression of spin-spin interaction (double resonance) and 2D-1H,1H-COSY experiments.

Preparation of F-S₁

Preparation of 3-aminopropyl 2-acetamido-2-deoxy-α-D-galactopyranosyl-(1→3)-β-D-galactopyranosyl-(1→4)-2-acetamido-2-deoxy-β-D-glucopyranoside (GalNAcα1-3Galβ1-4GlcNAc-S₁) (5) (SCHEME I)

The glycosyl chloride 3,4,6-tri-O-acetyl-2-azido-2-desoxy-β-D-galactopyranosylchloride (1) was prepared according to the method disclosed in the publication of Paulsen et al (1978). The glycosyl acceptor (3-trifluoroacetamidopropyl)-2-acetamido-3-O-acetyl-6-O-benzyl-2-deoxy-4-O-(2,4-di-O-acetyl-6-O-benzyl-β-D-galactopyranosyl)-β-D-glucopyranoside (2) was prepared according to the method disclosed in the publication of Pazynina et al (2008).

A solution of the glycosyl acceptor (420 mg, 0.5 mmol), silver triflate (257 mg, 1.0 mmol), tetramethylurea (120 μl, 1.0 mmol) and freshly calcinated

Substitute Specification (Clean) molecular sieves 4 Å in dry dichloromethane (20 ml), were stirred at room temperature in darkness for 30 min. Another portion of sieves 4 Å was added, and a solution of glycosyl chloride (350 mg, 1.0 mmol) in dry dichloromethane (3 ml) was added. The mixture was stirred for 20 h at room temperature. The resin was filtered and washed with methanol (4×10 ml), then solvent was evaporated. Chromatography on silica gel (elution with 5-7% isopropanol in chloroform) yielded 407 mg (70%) of the product 3 as a mixture of anomers (α/β=3.0 as determined by ¹H-NMR spectroscopy).

A solution of the product 3 (407 mg, 0.352 mmol) in methanol (30 ml) was subjected to hydrogenolysis over 400 mg 10% Pd/C for 16 h. Then the resin was filtered off, washed with methanol (4×10 ml) and the product concentrated in vacuum. The dry residue was acetylated with 2:1 pyridine-acetic anhydride mixture (6 ml) at 20° C. for 16 h, the reagents being co-evaporated with toluene. Two chromatography steps on silica gel (elution with 10% isopropanol in ethyl acetate and with 5-10% methanol in chloroform) resulted in 160 mg (42%) of the product 4 and 39 mg (10%) of the product 4p.

A solution of 2 M sodium methylate in methanol (200 μl) was added to a solution of the product 4 (160 mg, 0.149 mmol) in dry methanol (4 ml). The solution was evaporated after 1 h, 4 ml water added and the solution kept for 16 h before being chromatographed on a Dowex-H⁺ column (elution with 1 M ammonia). The eluate was evaporated, lyophilized to yield 87.2 mg (91%) of the 3-aminopropyltrisaccharide (5).

¹H NMR spectra were recorded on a Bruker BioSpin GmbH spectrometer at 303K. Chemical shifts (5) for characteristic protons are provided in ppm with the use of HOD (4.750), CHCl₃ (δ 7.270) as reference. Coupling constants (J) are provide in Hz. The signals in ¹H NMR spectra were assigned using a technique of spin-spin decoupling (double resonance) and 2D-¹H,¹H-COSY experiments.

The values of optical rotation were measured on a digital polarimeter Perkin Elmer 341 at 25° C.

Mass spectra were registered on a MALDI-TOF Vision-2000 spectrometer using dihydroxybenzoic acid as a matrix.

4: ¹H-NMR (700 MHz, CDCl₃): 1.759-1.834 (m, 1H, CH sp); 1.853-1.927 (m, 1H, CH sp); 1.972, 1.986, 1.996, 2.046, 2.053, 2.087, 2.106, 2.115, 2.130, 2.224 (10s, 10×3H, COCH₃); 3.222-3.276 (m, 1H, NCH sp); 3.544-3.583 (m, 1H, OCH sp); 3.591-3.661 (m, 2H, NCH sp, H-5a); 3.764 (dd ≈t, 1H, H-4a, J 8.8); 3.787 (dd, 1H, H-3b, J_(3,4) 3.7, J_(2,3) 9.9); 3.836 (br. t, 1H, H-5b, J 7.3); 3.882-3.920 (m, 1H, OCH sp) ; 3.950 (dd, 1H, H-6′c, J_(6′,6″) 10.6, J_(5,6′) 5.2); 4.009 (ddd, 1H, H-2a, J_(1,2) 7.9, J_(2,3) 10.0, J_(2,NH) 9.0); 4.076-4.188 (m, 5H, H-6′a, H-6′b, H-6″b, H-5c, H-6″c); 4.415 (d, 1H, H-1a, J_(1,2) 7.9); 4.443 (d, 1H, H-1b, J_(1,2) 7.9); 4.529 (dd, 1H, H-6″a, J_(6′,6″) 12.0, J_(5,6″) 2.5); 4.548 (ddd, 1H, H-2c, J_(1,2) 3.4, J_(2,3) 11.6, J_(2,NH) 9.4); 4.893 (dd, 1H, H-3c, J_(3,4) 3.1, J_(2,3) 11.6); 5.021 (d, 1H, H-1c, J_(1,2) 3.4); 5.039-5.075 (m, 2H, H-3a, H-2b); 5.339 (dd ≈d, 1H, H-4b, J 2.9); 5.359 (dd, 1H, H-4c, J_(3, 4) 2.7, J_(4, 5) 0.9); 5.810 (d, 1H, NHAc a, J_(2,NH) 9.0); 6.184 (d, 1H, NHAc c, J_(2,NH) 9.4); 7.310-7.413 (m, 1H, NHCOCF₃ sp). R_(f) 0.31 (EtOAc-iPrOH, 10:1). MS, m/z calculated for [C₄₃H₆₀N₃F₃O₂₅]H^(+:) 1076.35, found 1076.

4β: ¹H-NMR (700 MHz, CDCl₃): 1.766-1.832 (m, 1H, CH sp); 1.850-1.908 (m, 1H, CH sp); 1.923, 1.969, 1.982, 2.059, 2.071, 2.099 (2), 2.120, 2.136, 2.148 (10s, 10×3H, COCH₃); 3.230-3.289 (m, 1H, NCH sp); 3.521 (ddd, 1H, H⁻2c, J_(1,2) 8.2, J_(2,3) 11.2, J_(2,NH) 7.8); 3.548-3.591 (m, 1H, OCH sp); 3.591-3.648 (m, 2H, NCH sp, H-5a); 3.743 (dd≈t, 1H, H-4a, J 8.6); 3.795 (br. t, 1H, H-5b, J 6.5); 3.852 (dd, 1H, H-3b, J_(3,4) 3.6, J_(2,3) 9.9); 3.873-3.923 (m, 2H, H-5c, OCH sp); 4.002 (ddd, 1H, H-2a, J_(1,2) 8.0, J_(2,3) 9.5, J_(2,NH) 8.9); 4.039 (dd, 1H, H-6′b, J_(6′,6″) 11.6, J_(5,6) 6.9); 4.087-4.144 (m, 3H, H-6′a, H-6″b, H-6′c); 4.160 (dd, 1H, H-6″c, J_(6′,6″) 11.2, J_(5,6″) 6.0); 4.409, 4.417 (2d≈t, 2×1H, H-1a, H-1b, J 7.6); 4.519 (dd, 1H, H-6″a, J_(6″,6Δ) 11.8, J_(5,6″) 2.5); 4.992 (d, 1H, H-1c, J_(1,2) 8.2); 5.043 (dd, 1H, H-3a, J_(3,4) 8.6, J_(2,3) 9.5); 5.066 (dd, 1H, H-2b, J_(1,2) 8.0, J_(2,3) 9.8); 5.350 (dd≈d, 1H, H-4c, J 3.2); 5.372 (dd≈d, 1H, H-4b, J 3.4); 5.399 (d, 1H, NHAc c, J_(2,NH) 7.8); 5.449 (dd, 1H, H-3c, J_(3,4) 3.4, J_(2,3) 11.3); 5.856 (d, 1H, NHAc a, J_(2,NH) 8.9); 7.361-7.466 (m, 1H, NHCOCF₃ sp). R_(f) 0.24 (EtOAc-iPrOH, 10:1). MS, m/z calculated for [C₄₃H_(6C)N₃F₃O₂₅]H⁺:1076.35, found 1076.

5: ¹H-NMR (700 MHz, D₂O): 1.924-2.002 (m, 2H, CH₂ sp); 2.060, 2.064 (2s, 2×3H, NCOCH₃); 3.102 (m≈t, 2H, NCH₂ sp, J 6.8); 3.592-3.644 (m, 1H, H-5a); 3.655 (dd, 1H, H-2b, J_(1,2) 7.9, J_(2,3) 9.9); 3.702 (br. dd, 1H, H-5b, J_(5,6′) 3.8, J_(5,6″) 8.2, J_(4,5)≤1); 3.713-3.815 (m, 9H); 3.846 (dd, 1H, H-6′a, J_(6′,6″) 12.3, J_(5,6′) 5.3); 3.984-4.062 (m, 4H, OCH sp, H-6″a, H-4b, H-3c); 4.123 (dd≈d, 1H, H-4c, J 2.9); 4.206 (br. t, 1H, H-5c, J 6.3); 4.248 (dd, 1H, H-2c, J_(1,2) 3.6, J_(2,3) 11.0); 4.542 (2d≈t, 2H, H-la, H-lb, J 7.4); 5.100 (d, 1H, H-1c, J_(1,2) 3.5). Rf 0.55 (MeOH-1M aq. Py.AcOH, 5:1). MS, m/z calculated for [C₂₅H₄₅N₃O₁₆]H+: 644.28; found 644. [α]_(546 nm) +128 (c 0.3; MeCN—H₂O, 1:1).

5β: ¹H-NMR (700 MHz, D₂O): 1.938-1.991 (m, 2H, CH₂ sp); 2.055, 2.062 (2s, 2×3H, NCOCH₃); 3.100 (m≈t, 2H, NCH₂ sp, J 6.9); 3.610 (dd, 1H, H-2b, J₁,₂ 7.9, J_(2,3) 9.9); 3.603-3.636 (m, 1H, H-5a); 3.682 (br. dd, 1H, H-5b, J_(5,6′) 4.9, J_(5,6″) 7.8, J_(4,5)≤1); 3.693-3.826 (m, 11H); 3.842 (dd, 1H, H-6′a, J_(6′,6″) 12.1, J_(5,6′) 5.2); 3.934-3.972 (m, 2H, H-4b, H-2c); 4.012 (dd, 1H, H-6″a, J_(6′,6″) 12.2, J_(5,6″) 2.0); 4.023-4.057 (m, 1H, OCH sp); 4.175 (dd≈d, 1H, H-4c, J 2.9); 4.478 (d, 1H, H-1b, J1,2 7.9); 4.531 (d, 1H, H-1a, J_(1,2) 8.1); 4.638 (d, 1H, H-1c, J_(1,2) 8.4). R_(f) 0.48 (MeOH-1M aq. Py.AcOH, 5:1). MS, m/z calculated for [C₂₅H₄₅N₃O₁₆]H⁺: 644.28; found 644. [α] _(546 nm) +6 (c 0.3; MeCN-H₂O, 1:1).

Preparation of 3-aminopropyl-α-D-galactopyranosyl-(1→3)-β-D-galactopyranosyl-(1→4)-2-acetamido-2-deoxy-β-D-glucopyranoside (Galα1-3Galβ1-4GlcNAc-S₁; Galili-S₁) (9) (SCHEME II)

A mixture of the glycosyl acceptor 2 (500 mg, 0.59 mmol), thiogalactopyranoside 6 (576 mg, 1.18 mmol), NIS (267 mg, 1.18 mmol), anhydrous CH₂Cl₂ (25 ml) and molecular sieves 4 A (500 mg) was stirred at −45° C. for 30 min under an atmosphere of Ar. A solution of TfOH (21 μl, 0.236 mmol) in anhydrous CH₂Cl₂ (0.5 ml) was then added. The reaction mixture was stirred for 2 h at −45° C. and the temperature was then increased to −20° C. over 4 h. The mixture was kept at −20° C. overnight. Then extra amounts of thiogalactopyranoside 6 (144 mg, 0.295 mmol), NIS (66 mg, 0.295 mmol) and TfOH (5 μl, 0.06 mmol) were added and the stirring maintained at −20° C. for 2 h before being allowed to slowly warm up to r.t. (1 h). A saturated aqueous solution of Na₂S₂O₃ was then added and the mixture filtered. The filtrate was diluted with CHCl₃ (300 ml), washed with H₂O (2×100 ml), dried by filtration through cotton wool, and concentrated. Gel filtration on LH-20 (CHCl₃-MeOH) afforded the product 3-trifluoroacetamidopropyl-3,4-di-O-acetyl-2,6-di-O-benztk-β-D-galactopyranosyl-(1→3)-2,4-di-O-acetyl-6-O-benzyl-β-D-galactopyransoyl-(1→4)-2-acetamido-3-O-acetyl-6-O-benzyl-2-deoxy-β-D-glucopyranoside (7) (600 mg, 80%), as a white foam.

¹H NMR (700 MHz, CDCl₃, characteristic signals), δ, ppm: 1.78-1.82 (m, 4H, CHCHC, OC(O)CH₃), 1.84-1.90 (m, 1H, CHCHC), 1.91, 1.94, 1.97, 1.98, 2.06 (5 s, 5×3H, 4 OC(0)CH₃, NH(O)CH₃), 3.23-3.30(m, 1H, NCHH), 3.59-3.65 (m, 1H, NCHH), 4.05 (m, 1H, H-2^(I)), 4.33 (d, 1H, J,,₂ 7.55, H⁻1^(I)), 4.40 (d, 1H, J 12.04, PhCHH), 4.42 (d, 1H, J_(1,2) 8.07, H-^(II)), 4.45 (d, 1H, J 11.92, PhCHH) , 4.48 (d, 1H, J 12.00, PhCHH), 4.50 (d, 1H, J 12.00, PhCHH), 4.52 (d, 1H, J 12.04, PhCHH), 4.54 (d, 1H, J 12.00, PhCHH), 4.57 (d, 1H, J 12.00, PhCHH), 4.64(d, 1H, J 11.92, PhCHH), 4.99 (dd≈t, 1H, J 8.24, H-2^(II)), 5.08-5.13 (m, 2H, H-3^(I) , H-3^(III)), 5.23 (d, 1H, J_(1,2) 3.31, H⁻1^(III)), 5.46 (d, 1H, J_(3,4) 2.25, H-4^(II)), 5.54 (d, 1H, J_(3,4) 3.11, H-4^(III)), 7.20-7.40 (m, 20H, ArH); 7.49-7.54 (m, 1H, NHC(O)CF₃). R_(f) 0.4 (PhCH₃-AcOEt, 1:2).

The product 7 (252 mg, 0.198 mmol) was deacetylated according to Zemplen (8h, 40° C.), neutralized with AcOH and concentrated. The TLC (CH₃Cl-MeOH, 10:1) analysis of the obtained product showed two spots: the main spot with R_(f) 0.45, and another one on the start line (ninhydrin positive spot) that was an indication of partial loss of trifluoroacetyl. Therefore, the product was N-trifluorcacetylated by treatment with CF₃COOMe (0.1 ml) and Et₃N (0.01 ml) in MeOH (10 ml) for 1 h, concentrated and subjected to column chromatography on silica gel (CHCl₃-MeOH, 15:1) to afford the product 8 as a white foam (163 mg, 77%), R_(f) 0.45 (CH₃Cl-MeOH, 10:1). The product 8 was subjected to hydrogenolysis (200 mg Pd/C, 10 ml MeOH, 2 h), filtered, N-defluoroacetylated (5% Et₃N/ H₂O, 3 h) and concentrated. Cation-exchange chromatography on Dowex 50×4-400 (H⁺) (elution with 5% aqueous ammonia) gave the product 9 (90 mg, 98%) as a white foam.

¹H NMR (D₂O, characteristic signals), δ, ppm: 1.94-1.98 (m, 2H, CCH₂C), 2.07 (s, 3H, NHC(O)CH₃), 3.11 (m, J 6.92, 2H, NCH₂), 4.54 and 4.56 (2d, 2H, J_(1,2) 8.06, J_(1,2) 7.87, H-1^(T) and H-1^(TT)), 5.16 (d, 1H, J_(1,2) 3.87, H-1^(TTT)). R_(f) 0.3 (EtOH-BuOH-PY-H₂O-AcOH; 100:10:10:10:3).

Preparation of 3-aminopropyl α-D-galactopyranosyl-(1→4)-β-D-galactopyranosyl-(1→4)-β-D-glucopyranoside (21) and 2-aminoethyl α-D-galactopyranosyl-(1→4)-β-D-galactopyranosyl-(1→4)-β-glucopyranoside (28) (Galα1-4Galβ1-4Glc-S₁; Gb₃-S₁)

The title primary aminoalkyl variants of Gb₃-S₁, were prepared according SCHEME III, SCHEME IV and SCHEME V.

Preparation of (2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-(1→4)-2,3,6-tri-O-acetyl-α-D-glucopyranosyl trichloroacetimidate (10)

Trichloroacetonitrile (12.1 ml, 121 mmol) and DBU (0.45 ml, 3 mmol) were added to a solution of 10a (7.68 g, 12.1 mmol) in dry dichloromethane (150 ml) at −5° C. The reaction mixture was stirred at −5° C. for 3.5 h and concentrated in vacuo.

Flash chromatography (2:1 to 1:2 (0.1% Et₃N) toluene-ethyl acetate) of the residue provided 10 (6.01 g, 63.9%) as a light yellow foam, R_(f) 0.55 (2:1 toluene-acetone).

¹H NMR, CDCl₃: 1.95-2.2 (7s, 21H, 7Ac), 4.49 (d, 1H, J_(1,2=)8.07, H-1b), 4.91 (dd, 1H, J_(3,2)=10.3, J_(3,4)=2.8, H-3b), 5.05 (dd, 1H, J_(2,1)=3.5, J_(2,3)=9.3, H-2a), 5.12 (dd, 1H, J_(2,1)=8.07, J_(2,3)=10.3, H-2b), 5.32 (d, 1H, J_(4,3)=3, J_(4,5)<1, H-4b), 5.52 (t, 1H, J_(3,2)=J_(3,4)=9.29, H⁻3a), 6.48 (d, 1H, J_(1,2)=3.5, H-1a), 8.64 (s, 1H, HN=CCCl₃).

Preparation of 3-chloropropyl-(2,3,4,6-tetra-O-acetyl-(3-D-galactopyranosyl)-(1-4)-2,3,6-tri-O-acetyl-β-D-glucopyranoside (11)

A mixture of 2.94 g (3.8 mmol) of trichloroacetimidate 10, 0.66 ml (7.5 mmol) 3-chloropropanol, 50 ml dichloromethane, and 3 g of molecular sieves MS 4A was cooled to −5° C. An 8% solution of BF₃.Et₂O (0.4 mmol) in anhydrous dichloromethane was added drop wise with stirring.

After 30 min, the reaction mixture was filtered, diluted with chloroform (500 ml), and washed with water, saturated sodium hydrocarbonate solution, and water to pH 7. The washed reaction mixture was dried by filtration through a cotton layer and concentrated in vacuo.

Column chromatography on Silica gel (elution with 2.5:1 (v/v) toluene-ethyl acetate) resulted in 1.75 g (65%) of lactose derivative (11) as white foam. Rf 0.54 (2:1 toluene-acetone), Rf 0.50 (4:2:1 hexane-chloroform-isopropanol), [α]_(D) −4° (c 1.0, CHCl₃), m/z 712.2 (M⁺).

¹NMR, CDCl₃: 1.95 (br. s, 5H, Ac, —CH₂—), 2.0-2.2 (6s, 18H, 6Ac), 3.52 (m, 2H, —CH₂Cl), 3.63 (m, 1H, H-5a), 3.68 (m,1H, OCHH—), 3.79 (t, 1H, J=9.3, H-4a), 3.88 (m, 1H, H-5b), 3.93-3.98 (m, 1H, OCHH—), 4.05-4.15 (m, 3H, H-6a′, H-6b, H-6b′), 4.45 (d, 2H, H-1a, H-1b, J_(2,1)=7.83) 4.47 (m, 1H, H-6a), 4.89 (dd, 1H, J₂,₃=9.3, J_(2,1)=7.82, H-2a), 4.96 (dd, 1H, J_(3,2)=10.5, J_(3,4)=3.42, H-3b), 5.11 (dd, 1H, J_(2,3)=10.5, J_(2,1)=7.83, H-2b), 5.21 (t, 1H, J=9.3, H-3a), 5.35 (dd, 1H, J_(4,3)=3.42, J_(4,5)<1).

Preparation of 3-azidopropyl (2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-(1→4)-2,3,6-tri-O-acetyl-β-D-glucopyranoside (12)

A mixture of 2.15 g (3 mmol) of trichloropropylglycoside 11, 0.59 g (9 mmol) NaN₃, and 30 ml DMSO was maintained at 80° C. with stirring for 20 h. The mixture was then diluted with chloroform (500 ml), washed with water (4×100 ml), dried by filtration through a cotton layer, and concentrated in vacuo.

Column chromatography on Silica gel (elution with 8:2:1 hexane-chloroform-isopropanol) resulted in 1.96 g (91%) of glycoside (12) as a white foam, R_(f) 0.54 (2:1 (v/v) toluene-acetone), R_(f) 0.50 (4:2:1 (v/v/v) hexane-chloroform-isopropanol), isopropanol), [α]_(D) −5.4° (c 1.0, CHCl₃), m/z 718.8 (M⁺).

¹H NMR, CDCL₃: 1.85 (m, 2H, —CH ₂—), 1.98-2.2 (7s, 21H, 7Ac), 3.36 (m, 2H, —CH ₂N₃), 3.61 (m, 2H, H-5a, OCHH—CH₂—), 3.8 (t, 1H, J_(3,4)=J_(4,5)=9.29, H-4a), 3.85-3.94 (m, 2H, OCHH—CH₂; H-5b), 4.05-4.17 (m, 3H, H-6a, H-6a′, H-6b), 4.49 (d, 1H, J,,_(1,2)=8.07, H-1a), 4.5 (m, 1H, H-6b′), 4.51 (d, 1H, J,,₂=8.07, H-1b), 4.9 (dd, 1H, J_(1,2)=8.07, J_(2,3)=9.29, H-2a) , 4.97 (dd, 1H, J_(3,2)=10.27, J_(3,4)=3, H⁻3), 5.12 (dd, 1H, J_(2,1)=8.07, J_(2,3)=10.27, H-2b), 5.2 (t, 1H, J_(3,2)=J_(3,4)=9.29, H-3a), 5.36 (dd, 1H, J_(4,3)=3, J_(4,5)<1).

Preparation of 3-azidopropyl (4,6-O-benzylidene-β-D-galactopyranosyl)-(1-4)-β-D-glucopyranoside (13)

The lactoside 12 (1.74 g, 2.4 mmol) was deacetylated according to Zemplen and co-evaporated with toluene (2×30 ml). The residue was treated with α,α-dimethoxytoluene (0.65 ml, 3.6 mmol) and p-toluenesulfonic acid (50 mg, to pH 3) in DMF (20 ml) for 3 h. The reaction mixture was then quenched with pyridine, concentrated, and co-evaporated with o-xylene.

Column chromatography on Silica gel (elution with 9:1 (v/v) chloroform-isopropanol) and recrystalization (chloroform-methanol) resulted in 0.756 mg (62%) of benzylidene derivative (13). R_(f) 0.6 (5:1 chloroform-isopropanol), [α]_(D) −25.7° (c 1.0, methanol), m/z 513.4 (M⁺).

¹H NMR, CD₃OD: 2.06 (m, 2H, —CH ₂—), 3.45 (dd, 1H, J_(2,1)=J_(2,3)=9, H-2a) , 3.61 (m, 1H, H-5a), 3.64 (m, 2H, —CH ₂N₃), 3.74-3.9 (m, 6H, OCHH—; H-3a, H-4a; H-2b, H-3b, H-5b), 4.08-4.18 (m, 3H, H-6, H-6a′, OCHH—), 4.34-4.44 (m, 3H, H-6b, H-6b′, H-4b), 4.5 (d, 1H, J_(1,2)=7.9, H-1a), 4.68 (d, 1H, J_(1,2)=8, H-1b), 5.82 (s, 1H, CHPh), 7.55-7.72 (m, 5H, CHPh).

Preparation of 3-azidopropyl (4,6-O-benzylidene-3-O-benzyl-β-D-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-D-glucopyranoside (14)

Sodium hydride in mineral oil (290 mg, 12 mmol) was slowly added in 4 to 5 portions to a solution of 13 (726 mg, 1.5 mmol) in DMF (15 ml) at 0° C. with stirring. After 1 h, the ice bath was removed and benzyl bromide was added drop wise. The mixture was stirred overnight. 10 ml of methanol was then added. After 1 h, the mixture was diluted with chloroform (500 ml), and washed with water (3×200 ml), dried by filtration through a cotton layer, concentrated, and co-evaporated in vacuo with o-xylene.

Column chromatography on Silica gel (elution with 10:1 toluene-ethyl acetate) resulted in 1.24 g (87%) of lactose derivative 14 as white foam, R_(f) 0.56 (5:3 (v/v) hexane-ethyl acetate), [α]_(D) +10.8° (c 1.0, CHCl₃), m/z 963.8 (M⁺).

¹H NMR, CDCl₃: 1.85 (m, 2H, —CH ₂—), 2.91 (m, 1H, H-5b), 3.33 (m, 1H, H-5a), 3.34-3.42 (m, 4H, H-2a, H-3b, —CH ₂N₃), 3.55-3.62 (m, 2H, OCHH—; H-3a), 3.73 (dd, 1H, J_(2,1)=8, J_(2,3)=10, H-2b), 3.92-3.97 (m, 2H, H-4a, OCHH—), 4.0 (br. d, 1H, J_(4,3)=3.6, H-4b), 4.34 (d, 1H, J_(1,2)=7.9, H-1a), 4.42 (d, 1H, J_(1,2)=8, H-1b), 5.43 (s, 1H, CH(Bd), 7.14-7.50 (m, 30H, Ph).

Preparation of 3-azidopropyl (2,3,6-O-tri-O-benzyl-β-D-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-D-glucopyranoside (15)

Hydrogen chloride in diethyl ether was added to a mixture of 14 (1.24 g, 1.3 mmol), sodium cyanoborohydride (0.57 g, 9.1 mmol), and freshly activated molecular sieves MS 3 Å (33 g) in anhydrous THF (20 ml) until the evolution of gas ceased.

The mixture was stirred for 2 h, diluted with chloroform (300 ml), washed with water, saturated sodium hydrocarbonate solution, and water to pH 7. The washed mixture was dried by filtration through a cotton layer and concentrated in vacuo.

Column chromatography on Silica gel (elution with 20:1 to 7:3 (v/v) toluene-ethyl acetate) resulted in 0.91 g (65%) of lactose derivative 15 as a white foam, R_(f) 0.42 (9:1 (v/v) toluene-acetone), [α]_(D)+17.8° (c 1.0, CHCl₃), m/z 965.8 (M⁺).

¹H NMR, CDCl₃: 1.85 (m, 2H, —CH ₂—), 2.39 (d, 1H, J=2.2, OH), 4.04 (br. s, 1H, H-4b), 4.34 (d, 1H, J_(1,2)=7.9, H-1a), 4.42 (d, 1H, J_(1,2)=8, H-lb), 7.14-7.50 (m, 30H, Ph).

¹H NMR of acetylated analytical probe 15a, CDCl₃: 1.85 (m, 2H, —CH ₂—), 4.34 (d, 1H, J_(1,2)=7.9, H-1a), 4.42 (d, 1H, J_(1,2)=8, H-1b), 5.5 (br. d, 1H, J_(4,3)=3.43, H-4b), 7.14-7.50 (m, 30H, Ph).

Preparation of 3-trifluoroacetamidopropyl (2,3,6-O-tri-O-benzyl-β-D-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-D-glucopyranoside (16)

A mixture of derivative 15 (0.914 g, 0.94 mmol), triphenylphosphine (0.5 g, 1.9 mmol) and THF (10 ml) was stirred for 0.5 h, 100 μl of water added, and the mixture stirred overnight. The reaction mixture was then concentrated and co-evaporated with methanol. The residue was dissolved in methanol (15 ml) and triethylamine (30 μl) and methyl trifluoroacetate (0.48 ml, 4.7 mmol) added. The solution was held for 30 min and then concentrated.

Column chromatography on Silica gel (elution with 5:1 to 1:1 (v/v) hexane-acetone) acetone) resulted in 0.87 g (84%) of lactose derivative 16 as white foam, R_(f) 0.49 (9:1 (v/v) hexane-acetone), [α]_(D) +17° (c 1.0, CHCl₃), m/z 1060.1 (M⁺+Na).

¹H NMR, CDCl₃: 1.88 (m, 2H, —CH ₂—), 2.40 (br. s, 1H, OH), 4.05 (br. s, 1H, H-4b), 4.36 (d, 1H, J_(1,2) =7.8, H-1a), 4.40 (d, 1H, J_(1,2) =7.6, H-1b), 7.10-7.35 (m, 30H, Ph).

Preparation of 2,3,4,6-tetra-O-benzyl-β-D-galactopyranosyl trichloroacetimidate (18)

A mixture of galactose derivative 17 (2 g, 3.65 mmol), trichloroacetonitrile (1.75 ml, 17.55 mmol), anhydrous potassium carbonate (2 g, 14.6 mmol), and dichloromethane (4 ml) was stirred for 22 h at room temperature under argon.

The mixture was then filtered through a Celite layer and concentrated in vacuo. Column chromatography on Silica gel (elution with 4:1 (v/v) hexane-ethyl acetate (1% Et₃N) resulted in 1.5 g (60%) of 18 as white foam, R_(f) 0.47 (7:3 (v/v) hexane-ethyl acetate containing 1% Et₃N) and 0.46 g (0.8 mmol, 23%) of the starting derivative 17, R_(f) 0.27 (7:3 (v/v) hexane-ethyl acetate containing 1% Et₃N).

¹H NMR (CDC1₃): 3.60-3.70 (m, 3H, H-3, H-6, H-6′), 3.75 (t, 1H, J_(5,6)=6.30, H-5), 3.98 (d, 1H, J_(4,36)=2.19, H-4), 4.08 (dd, 1H, J_(2,3)=9.73 , J_(2,1)=7.95, H-2), 4.42 and 4.47 (ABq, 2H, J=12.00, PhCH ₂), 4.63 and 4.95 (ABq, 2H, J=11.51, PhCH2), 4.72 (s, 2H, PhCH ₂), 4.80 and 4.90 (ABq, 2H, J=10.95, PhCH ₂), 5.74 (d, 1H, J_(1,2)=7.95, H-1), 7.22-7.35 (m, 20H, ArH), 8.62 (s, 1H, NH).

Preparation of 3-trifluoroacetamidopropyl (2,3,4,6-tetra-O-benzyl-α-D-galactopyranosyl)-(1→4)-(2,3,6-tri-O-benzyl-β-D-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-D-glucopyranoside (19)

A mixture of lactose derivative 16 (158 mg, 0.153 mmol), trichlroroacetimidate 18 (120 mg, 0.175 mmol), molecular sieves MS 4 Å (0.5 g), and dichloromethane (5 ml) was stirred for 30 min at room temperature under argon. 0.1 ml of a 1% (v/v) solution of trimethylsilyl trifluoromethanesulfonate in dichloromethane was then added. After 2 h, another 50 mg (0.073 mmol) trichlroroacetimidate 18 and 30 μl of a 1% (v/v) solution of trimethylsilyl trifluoromethanesulfonate in dichloromethane were added. The reaction mixture was stirred overnight at +4 ° C., quenched with triethylamine (5 μl), filtered, and concentrated in vacuo.

Column chromatography on Silica gel (elution with 12:1 to 1:1 (v/v) toluene-ethyl acetate) resulted in 170 mg (72%) of trisaccharide 19; R_(f) 0.56 (4:1 (v/v) toluene-ethyl acetate); [α]_(D) +30.8° (c 1.0, CHCl₃).

¹H NMR, CDCl₃: 1.78-1.89 (m, 2H, —CH ₂—), 4.34 (d, 1H, J_(1,2)=7.8, H-1a), 4.43 (d, 1H, J_(1,2)=7.4, H-1b), 5.06 (d, 1H, J_(1,2)=3.0, H-1c), 7.14-7.48 (m, 50H, Ph).

Preparation of 3-trifluoroacetamidopropyl (2,3,4,6-tetra-O-acetyl-α-D-galactopyranosyl)-(1 ,4)-(2,3,6-tri-O-acetyl-β-D-galactopyranosyl)-(1 ,4)-2,3,6-tri-O-acetyl-β-D-glucopyranoside (20)

The catalyst 10% Pd/C (10 mg) was added to a solution of the protected oligosaccharide 19 (73 mg, 0.047 mmol) in methanol (7 ml), the mixture degassed, and the flask filled with hydrogen. The reaction mixture was stirred for 1 h, filtered off from the catalyst through a Celite layer, and concentrated in vacuo. The dry residue was dissolved in pyridine (2 ml), acetic anhydride (1 ml) added, and the mixture held for 3 h. The solvents were then evaporated and residue co-evaporated with toluene (4×2 ml).

Column chromatography on Silica gel (elution with 2:1 hexane-acetone) resulted in 43.5 mg (90%) of trisaccharide 20 as a white foam, R_(f) 0.52 (2:1 hexane-acetone), [α]_(D) +30.4° (c 1.0, CHCl₃).

¹H NMR, CDCl₃: 1.87 (2H, m, CH₂); 1.99, 2.05, 2.05, 2.06, 2.07, 2.07, 2.09, 2.09, 2.12, and 2.14 (10×3H, 10 s, 10 Ac); 3.37 and 3.52 (2×1H, 2 m, 2 CHN); 3.63 (1H, ddd, J_(4,5)=9.8, J_(5,6)=4.9, J_(5,6)=2.0, H-5a); 3.72 (1H, m, OCH); 3.77 (1H, ddd≈br. T, J_(4,5)<1, J5,6=6.8, J5,6=6.1, H-5b); 3.79 (1H, dd, J_(3,4)=9.3, J_(4,5)=9.8, H-4a); 3.87 (1H, m, OCH); 4.02 (1H, dd≈br. d, J_(3,4)=2.5, J_(4,5)<1, H-4b); 4.09 (1H, dd, J_(5,6)=4.9, J_(6,6)=12.0, H-6a); 4.12 (1H, dd, J_(5,6)=5.6, J_(6,6′) =10.8, H-6c); 4.14 (1H, dd, J_(5,6)=6.8, J_(6,6′) =11.0, H-6b); 4.17 (1H, dd, J_(5,6′) =8.6, J_(6,6′) =10.8, H-6′c); 4.45 (1H, dd, J_(5,6′) =6.1, J_(6,6′) =11.0, H-6′b); 4.49 (1H, ddd □ br. T, J_(4,5)<1, J_(5,6)=5.6, J_(5,6)=8.6, H-5c); 4.50 (1H, d, J_(1,2)=7.8, H-1a); 4.55 (1H, d, J_(1,2)=7.8, H-1b); 4.59 (1H, dd, J_(5,6′) =2.0, J_(6,6′)=12.0, H-6′a); 4.76 (1H, dd, J_(2,3)=10.8, J_(3,4)=2.5, H-3b); 4.86 (1H, dd, J_(1,2)=8.1, J_(2,3)=9.5, H-2a); 4.10 (1H, d, J_(1,2)=3.4, H-1c); 5.12 (1H, dd, J_(1,2)=7.8, J_(2,3)=10.8, H-2b); 5.19 (1H, dd, J_(1,2)=3.4, J_(2,3)=11.0, H-2c); 5.22 (1H, dd≈T, J_(2,3)=9.5, J_(3,4)=9.3, H-3a); 5.40 (1H, dd, J_(2,3)=11.0, J_(3,4)=3.4, H-3c); 5.59 (1H, dd≈br. d, J_(3,4)=3.4, J_(4,5)<1, H-4c); 7.09 (1H, m, NHCOCF₃).

Preparation of 3-aminopropyl α-D-galactopyranosyl-(1→4)-β-D-galactopyranosyl-(1→4)-β-D-glucopyranoside (Gb₃-S₁) (21)

Sodium methylate (30 μl of 2 M solution in methanol) was added to a solution of trisaccharide (20) (43 mg, 0.042 mmol) in anhydrous methanol (3 ml) and held for 2 h. The solution was then concentrated in vacuo, water (3 ml) added, and the mixture held for 3 h. The mixture was then applied to a column (10×50 mm) with Dowex 50×4-400 (H⁺) cation exchange resin.

The target compound was eluted with 1 M aqueous ammonia and the eluant concentrated in vacuo. Lyophilization from water provided trisaccharide 21 (23 mg, quant.) as a colorless powder. R_(f) 0.3 (100:10:10:10:2 (v/v/v/v/v) ethanol-n-butanol-pyridine-water-acetic acid), [α]_(D) +42° (c 1; water), m/z 584.9 (M⁺+Na).

¹H NMR, D₂O: 1.98-2.05 (m, 2H, —CH ₂—), 3.17 (m, 2H, —CH ₂NH₂), 3.33-3.35 (m, 1H, H-2a), 4.36 (m, 1H, H-5c), 4.53 (d, 2H, J=7.8, H-1a, H-1b), 4.97 (d, 1H, J_(1,2)=3.67, H-1c).

Preparation of 2-azidoethyl (3,4-di-O-acetyl-2,6-di-O-benzyl-α-D-galactopyranosyl)-(1→4)-(2,3,6-tri-O-benzyl-β-D-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-D-glucopyranoside (25)

To the solution of ethyl 3,4-di-O-acetyl-2,6-di-O-benzyl-1-thio-β-D-galactopyranoside (23) (550 mg, 1.11 mmol) in dichloromethane (10 ml) was added Br₂ (57 μl, 1.11 mmol). The mixture was held for 20 min at room temperature, then concentrated in vacuo at room temperature and co-evaporated with anhydrous benzene (3×30 ml). The crude 3,4-di-O-acetyl-2,6-di-O-benzyl-α-D-galactopyranosylbromide (24) was used for glycosylation without purification.

The mixture of lactose derivative 22 (Sun et al (2006)) (500 mg, 0.525 mmol), 1,1,3,3-tetramethylurea (300 pl), molecular sieves MS 4 Å (1 g), and dichloromethane (25 ml) was stirred for 30 min at room temperature. Silver trifluoromethanesulfonate (285 mg, 1.11 mmol), molecular sieves MS 4 Å (0.5 g), and the freshly prepared galactopyranosylbromide (24) in dichloromethane (15 ml) were then added. The reaction mixture was stirred overnight, filtered, and concentrated in vacuo.

Column chromatography on Silica gel (elution with 3:1 to 1:1 (v/v) hexane-ethyl acetate) resulted in 570 mg (79%) of trisaccharide 25, Rf 0.25 (2:1(v/v) hexane-ethyl acetate); [α]_(D) +32° (c 0.8, CHCl₃)

¹H NMR, CDCl₃: 1.88, 1.94 (2s, 2Ac) , 3.00 (dd, 1H, J_(5,6)=4.9, J_(6′,6)=8.4, H-6a), 3.19 (dd, J_(1,2)=8.5, J_(2,3)=8.9, H-2a), 3.30-3.36 (m, 2H, —CHHN₃, H-6′a), 3.38-3.47 (m, 4H, H-5a, H-5b, H-2b, H-6b), 3.48-3.54 (m, 1H, —CHHN₃), 3.61 (dd, 1H, J_(2,3)=8.9, J_(3,4)=9.2, H-3a), 3.69-3.75 (m, 3H, H-6′b, H-6c, —OCHH—), 3.85 (dd, 1H, J_(5,6)=4.6, J_(6,6′)=11.0, H-6c), 3.89 (dd, 1H, J_(1,2)=3.4, J_(2,3)=10.8, H-2c), 3.95 (dd, 1H, J_(3,4)=9.2, J_(4,5)=9.5, H-4a), 4.0-4.1 (m, 4H, —OCHH—, H-4b, CH₂Ph), 4.25, 4.29, 4.32, 4.39 (4 d, 4×1H, J,_(AB)=12, 4 —CHPh), 4.43 (d, 1H, J,,_(1,2)=7.6, H-1), 4.48 (d, 1H, J_(1,2)=7.6, H-1), 4.54-4.62 (m, 5H, 4 —CHPh, H-5c), 4.71-4.84 (m, 4H, 4 —CHPh), 4.89, 4.91, and 5.09 (3 d, 3×H, 3 4 —CHPh), 5.15 (d, 1H, J_(1,2)=3.0, H-1c), 5.39 (dd, 1H, J_(2,3)=10.8, J_(3,4)=3.4, H-3c), 5.56 (dd, 1H, J_(3,4)=3.4, J_(4,5)=0.9, H-4c), 7.14-7.48 (m, 40H, Ph).

Preparation of 2-aminoethyl α-D-galactopyranosyl-(1→4)-β-D-galactopyranosyl-(1→4)-β-D-glucopyranoside (Gb₃-S₁) (28)

Sodium methylate (100 μl of 2 M solution in methanol) was added to a suspension of trisaccharide (25) (500 mg, 0.363 mmol) in anhydrous methanol (50 ml). The mixture was stirred overnight at room temperature, quenched with acetic acid, and concentrated in vacuo.

Column chromatography on Silica gel (elution with 2:1 to 1:1 (v/v) hexane-ethyl acetate) resulted in 470 mg of trisaccharide (26), R_(f) 0.5 (1:1 (v/v) hexane-ethyl acetate), [α]_(D) +36° (c 0.5, CHCl₃).

To a solution of trisaccharide (26) and Boc₂O ((150 mg, 0.91 mmol) in anhydrous methanol (50 ml) was added the catalyst 10% Pd/C (500 mg). The mixture was degassed and the flask filled with hydrogen. The reaction mixture was stirred for 3 h, filtered off from the Pd/C, and concentrated in vacuo.

Column chromatography on Silica gel (elution with 6:5:1 (v/v/v) chloroform-ethanol-water) resulted in 160 mg (68%) of trisaccharide 27 R_(f) 0.3 (6:5:1 (v/v/v) dichloromethane-ethanol-water). ¹NMR, D₂O: 1.45 (s, 9H, (CH₃)₃COCO-13 ), 4.53 (d, 1H, J_(1,2)=7.8, H-1b), 4.58 (d, 1H, J_(1,2)=7.4, H-1b), 4.98 (d, 1H, J_(1,2)=3.0, H-1c).

The trisaccharide 27 was then treated with 95% CF₃COOH (5 ml, 10 min). Upon completion, the mixture was concentrated in vacuo, co-evaporated with toluene, and applied to a column (10×100mm) of Dowex 50×4-400 (H⁺) cation exchange resin. The target compound was eluted with 1 M aqueous ammonia and the eluant was concentrated in vacuo. Lyophilization from water provided trisaccharide 28 (135, quant.) as a colorless powder. R_(f) 0.35 (100:10:10:10:2 (v/v/v/v/v) ethanol-n-butanol-pyridine-water-acetic acid), [α]_(D) +25° (c 0.2; water).

¹H NMR, D₂O:: 3.32 (m, 2H, —CH₂NH₂), 3.40-3.45 (m, 1H, H-2a), 3.63 (dd, 1H, J_(1,2)=7.9, J_(2,3)=10.3, H-2b), 3.66-3.78 (m, 5H, H-5a, H-3a, H-4a, H-6c, H-6′c), 3.8 (dd, 1H, J_(3,4)=3.1, J_(3,2)=10.3, H-3b), 3.84 (m, 2H, J_(5,6)=4.4, J_(5,6′)=7.9, H-5b), 3.88-3.92 (m, 3H, H-2c, H-6b, —OCHH—), 3.96 (dd, 1H, J_(3,4)=3.3, J_(3,2)=10.3, H-3c), 3.98-4.03 (m, 2H, H-6a, H-6′b), 4.06 (dd, 1H, J_(5,6)=2.2, J_(6,6′)=12.3, H-6′a), 4.08 (dd, 1H, J_(3,4)=3.3, J_(4,5)=0.9, H-4c), 4.09 (d, 1H, J_(3,4)=3.1, H-4b) , 4.17-4.21 (m, 1H, —OCHH—), 4.41 (m, 1H, H-5c), 4.56 (d, 1H, J=7.9, H-1b), 4.60 (d, 1H, J=8.1, H-1a), 5.00 (d, 1H, J_(1,2)=3.9, H-1c).

The preparation of the primary amino propyl glycosides GalNAcα1-3(Fucα1-2)Galβ-O(CH₂)₃NH₂ (A_(tri)-S₁) and Galα1-3(Fucα1-2)Galβ-O(CH₂)₃NH₂ (B_(tri)-S₁) is described in the publication of Korchagina and Bovin (1992). The preparation of the primary amino propyl glycosides Galα1-3(Fucα1-2)Galβ1-3GlcNAcβ-O (CH₂)₃NH₂ (B_(tetra)(Type 1)—S₁), Galα1-3(Fucα1-2)Galβ1-3GlcNAcα-O (CH₂)₃NH₂ (B_(tetra) (Type 3)-S₁ and Galα1-3(Fucα1-2)Galβ1-3GalNAcβ-O(CH₂) ₃NH₂ (B_(tetra)(Type 4)-S₁) is described in the publication of Korchagina et al (2009). The preparation of the primary amino propyl glycosides Xylα1-3Glcβ-O(CH₂)₃NH₂ and Xylα1-3Xylα1-3Glcβ-O(CH₂)₃NH₂ is described in the publication of Krylov et al (2007). The preparation of the primary amino propyl glycosides Neu5Acα2-3Galβ1-3(Fucα1-4)GlcNAcβ-O(CH₂)₃NH₂ (sLe^(a)-S₁) and Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAcβ-O(CH₂)₃NH₂ (sLe^(x)-S₁) is described in the publication of Nifant'ev et al (1996). The preparation of the primary amino propyl glycosides GalNAcβ1-3(Fucα1-2)Galβ1-4GlcNAcβ-O(CH₂)₃NH₂ (A_(tetra)(Type 2)-S₁), Galα1-3(Fucα1-2)Galβ1-4GlcNAcβ-O(CH₂)₃NH₂ (B_(tetra) (Type 2)-S₁) Fucα1-2Galβ1-4GlcNAcβ-O(CH₂)₃NH₂ (H_(tri) (Type 2)-S₁), Galβ1-4(Fucα1-3)GlcNAcβ-O(CH₂)₃NH₂ (Le^(x)-S₁), Fucαl-2Galβ1-4(Fucα1-3)GlcNAcβ-O(CH₂)₃NH₂ (Le^(y)-S₁), Galα1-3Galβ1-4GlcNAcβ-O(CH₂) ₃NH₂ (B_(tri)(Type 2) —S₁) and Galα1-4Galβ1-4GlcNAcβ-O(CH₂)₃NH₂ (P₁-S₁) is decribed in the publication of Pazynina et al (2002). The preparation of the primary amino propyl glycosides Neu5Acα2-3Galβ-O(CH₂)₃NH_(2,) Neu5Acα2-3Galβ1-4GlcNAcβ-O(CH₂)₃NH₂ (3′SLN-S₁), Neu5Acα2-3Galβ1-4(6-HSO₃)GlcNAcβ-O(CH₂)₃NH₂ (6-Su-3′SLN-S₁), Neu5Acα2-3Galβ1-3GalNAcα-O(CH₂)₃NH₂ (SiaTF-S₁), Neu5Acα2-3Galβ1-3(6-HSO₃)GalNAcα-O(CH₂)₃NH₂ (6-Su-SiaTF-S₁), Neu5Acα2-3Galβ1-3GlcNAcβ-O(CH₂)₃NH₂ (SiaLe^(c)-S₁), Neu5Acα2-3Galβ1-3(6-HSO₃)GlcNAcβ-O(CH₂) _(3NH) ₂ (6-Su-SiaLe^(c)-S₁), Neu5Acα2-3Galβ1-3(Fucα1-4)GlNAcβ-O (CH₂)₃NH₂ (SiaLe^(a)-S₁), Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAcβ-O(CH₂)₃NH₂ (SiaLe^(x)-S₁) is decribed in the publication of Pazynina et al (2003). The preparation of the primary amino propyl glycoside Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ-O(CH₂)₃NH₂ (trilactosamine-S₁) is described in the publication of Pazynina et al (2008). The preparation of the primary amino propyl glycosides GalNAca1-3(Fucα1-2)Galβ1-3GlcNAcβ-O(CH₂)₃NH₂ (A_(tetra)(Type 1)-S₁), GalNAcα1-3(Fucα1-2)Galβ1-3GlcNAcα-O(CH₂)₃NH₂ (A_(tetra)(Type 3) —S₁) and GalNAcα1-3(Fucα1-2)Galβ1-3GalNAcβ-O(CH₂)₃NH₂ (_(A) _(tetra)(Type 4) -S₁) is described in the publication of Ryzhov et al (2012). The preparation of a number of primary aminoethyl mono- and disaccharides is described in the publication of Sardzik et al (2010). The preparation of the primary amino propyl glycosides Neu5Ac-α-(2-6′)-Galβ1-4GlcNAcβ-O(CH₂)₃NH₂ (Neu5Ac-α-(2-6′)-lactosamine-S₁) and Neu5Gc-α-(2-6′)-Galβ1-4GlcNAcβ-O(CH₂)₃NH₂ (Neu5Gc-α-(2-6′)-lactosamine-S₁) is described in the publication of Sherman et al (2001). The preparation of primary amino propyl glycosides of linear β-(1-3)-D-glucooloigosaccharides containing from 3 to 13 monosaccharide units is described in the publication of Yashunsky et al (2016).

Several fluorescent compounds are available commercially and may be used in the preparation of synthetic molecule constructs. For example, fluorescein isothiocyanate (FITC) may be conjugated with with a diamine such as 1,5-diaminopentyl (cadaverine) and dipyrrometheneboron difluoride (BODIPY™) may be conjugated with an alkionyl diamine such as propionly ethylenediamine (BODIPY™ FL EDA). Synthetic molecule constructs where F is one of the following fluorophores (represented as neutrally charged protonated species) may thereby be prepared:

Preparation of S₂-L

Preparation of Activated 1,2-O-dioleoyl-sn-glycero-3-phosphatidylethanolamine (SuNO-Ad-DOPE; S₂-L) (31) (SCHEME VIA)

To a solution of bis(N-hydroxysuccinimidyl) adipate (29) (70 mg, 205 μmol) in dry N,N-dimethylformamide (1.5 ml), 1,2-O-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE)(30) (40 μmol) in chloroform (1.5 ml) was added, followed by triethylamine (7 μl). The mixture was kept for 2 h at room temperature, then neutralized with acetic acid and partially concentrated under vacuum. Column chromatography (Sephadex LH-20, 1:1 chloroform-methanol, 0.2% acetic acid) of the residue yielded the the activated lipid 31 (37 mg, 95%) as a colorless syrup. TLC (chloroform-methanol-water, 6:3:0.5): R_(f) =0.5 31: ¹H NMR (CDCl₃/CD₃OD, 2:1) 5.5 (m, 4H, 2×(—CH=CH—), 5.39 (m, 1H, —OCH₂-CHO—CH₂O—), 4.58 (dd, 1H, J=3.67, J=11.98, -CCOOHCH—CHO-CH₂O—), 4.34 (dd, 1H, J=6.61, J=11.98, —CCOOHCH—CHO—CH₂O—), 4.26 (m, 2H, PO—CH₂ —CH₂—NH₂), 4.18 (m, 2H, —CH₂ —OP), 3,62 (m, 2H, PO—CH₂—CH₂ —NH₂), 3.00 (s, 4H, ONSuc), 2.8 (m, 2H, —CH₂ —CO (Ad), 2.50 (m, 4H, 2×(—CH₂ —CO), 2.42 (m, 2H, —CH₂ -CO (Ad), 2.17 (m, 8H, 2×(—CH₂ —CH=CH—CH₂ —), 1.93 (m, 4H, COCH₂CH₂ CH₂ CH₂CO), 1.78 (m, 4H, 2×(COCH₂CH₂ —), 1,43, 1.47 (2 bs, 40H, 20 CH2), 1.04 (m, 6H, 2 CH3).

Preparation of activated 1,2-O-distereoyl-sn-glycero-3-phosphatidylethanolamine (SuNO-Ad-DSPE; S₂-L) (33) (SCHEME VIB)

To a solution of bis(N-hydroxysuccinimidyl) adipate (29) (70 mg, 205 pmol) in dry N,N-dimethylformamide (1.5 ml) was added 1,2-O-distearyl-sn-glycero-3-phosphatidylethanolamine (DSPE)(32) (40 pmol) in chloroform (1.5 ml) followed by triethylamine (7 pl). The mixture was kept for 2 h at room temperature, then neutralized with acetic acid and partially concentrated in vacuo. Column chromatography (Sephadex LH-20, 1:1 chloroform-methanol, 0.2% acetic acid) of the residue yielded the activated lipid 33 (37 mg, 95%) as a colorless syrup; TLC (chloroform-methanol-water, 6:3:0.5): R_(f) =0.55.

33: ¹H NMR (CDCl₃/CD₃OD, 2:1), 5.39 (m, 1H, —OCH₂—CHO—CH₂O—), 4.53 (dd, 1H, J=3.42, J=11.98, —CCOOHCH—CHO—CH₂O—), 4.33 (dd, 1H, J=6.87, J=11.98, —CCOOHCH—CHO—CH₂O—), 4.23 (m, 2H, PO—CH₂—CH₂—NH₂), 4.15 (m, 2H, —CH₂—OP) , 3,61 (m, 2H, PO—CH₂—CH₂—NH₂), 3.00 (s, 4H, ONSuc), 2.81 (m, 2H, —CH₂—CO (Ad), 2.48 (m, 4H, 2×(—CH₂—CO), 2.42 (m, 2H, —CH₂—CO (Ad), 1.93 (m, 4H, COCH₂CH₂CH₂CH₂CO), 1.78 (m, 4H, 2×(COCH₂CH₂—), 1,43, 1.47 (2 bs, 40H, 20CH2), 1.04 (m, 6H, 2CH3).

Preparation of F—S₁-S₂-L Condensing Activated DOPE (or DSPE) with Aminopropylglycoside.

To a solution of activated DOPE (or DSPE) (A-L) (33 μmol) in N,N-dimethylformamide (1 ml) 30 μmol of Sug-S₁—NH₂ (F—S₁—NH₂) and 5 μl of triethylamine were added. For example, the Sug may be either the aminopropyl glycoside (F—S₁—NH₂) of either GalNAcα1-3(Fucα1-2)Galβ trisaccharide (A-glycotope) (F) or Galα1-3(Fucα1-2)Galβtrisaccharide (B-glycotope) (F).

The mixture was stirred for 2 h at room temperature. Column chromatography (Sephadex LH-20 in 1:1 chloroform-methanol followed by silica gel in ethyl acetate-isopropanol-water, 4:3:1 (v/v/v) of the mixture typically yielded 85-90% of the synthetic molecule construct, for example, A_(tri)-sp-Ad-DOPE (I) or B_(tri)-sp-Ad-DOPE (VI).

¹H NMR (CDCl₃/CD₃OD, 1:1), δ:

A_(tri)-sp-Ad-DOPE (I)—5.5 (m, 4H, 2×(—CH=CH—), 5.43-5,37 (m, 2H, H-1 (GalNHAc) and —OCH₂—CHO—CH₂O—), 5.32 (d, 1H, H-1, J-3.5 H-1 Fuc), 2.50 (m, 4H, 2×(—CH₂ —CO), 2.40 (m, 4H, COCH₂ CH₂CH₂CH₂ CO), 2.20 (m, 8H, 2×(—CH₂ —CH=CH—CH₂ —), 2.1 (s, 3H, NHAc), 1.92 (m, 2H, O—CH₂CH₂ CH₂—NH), 1.8 (m, 8H, COCH₂CH₂ CH₂ CH₂CO and 2×(COCH₂CH₂ ), 1.43, 1.47 (2 bs, 40H, 20 CH₂), 1.40 (d, 3H, J=6.6, CH₃ Fuc), 1.05 (m, 6H, 2 CH₃).

A_(tri)-spsp₁-Ad-DOPE (II)—5.5 (m, 4H, 2×(—CH=CH—), 5.43-5.37 [m, 2H, H-1 (GalNHAc) and —OCH₂—CHO—CH₂O—], 5.32 (d, 1H, H-1, J=3.6 H-1 Fuc), 2.50 (m, 4H, 2×(—CH₂ —CO), 2.40-2.32 (m, 6H, COCH₂ CH₂CH₂CH₂ CO and COCH₂ — (sp₁), 2.18 [m, 8H, 2×(—CH₂ —CH=CH—CH₂ —)], 2.1 (s, 3H, NHAc), 1.95(m, 2H, O—CH₂CH₂ CH₂—NH), 1.8 [m, 10H, COCH₂CH₂ CH₂ CH₂CO, 2×(COCH₂CH₂ — . . . ), —COCH₂CH₂ (CH₂)₃NH—], 1.68 (m, 2H, CO(CH₂)₃CH₂ CH₂NH—), 1,43, 1.47 (2 bs, 42H, 22 CH₂), 1.37 (d, 3H, J=5.6, CH₃ Fuc), 1.05 (m, 6H, 2 CH₃).

A_(tri)-sp-Ad-DSPE (III)—5.42-5.38 (m, 2H, H-1 (GalNHAc) and —OCH₂—CHO—CH₂O—), 5.31 (d, 1H, H-1, J=3.5 H-1 Fuc), 2.48 [m, 4H, 2×(—CH₂ —CO)], 2.42 (m, 4H, COCH₂ CH₂CH₂CH₂ CO), 2.18 (s, 3H, NHAc), 1.95 (m, 2H, O—CH₂CH₂ CH₂—NH), 1.8 [m, 8H, COCH₂CH₂ CH₂ CH₂CO and 2×(COCH₂CH₂ —)], 1.43, 1.47 (2 bs, 56H, 28 CH₂), 1.38 (d, 3H, J=6.6, CH₃ Fuc) , 1.05 (m, 6H, 2 CH₃).

B_(tri)-sp-Ad-DOPE (VI)—5.5 (m, 4H, 2×(—CH=CH—), 5.42-5,38 [m, 2H, H-1 (Gal) and —OCH₂—CHO—CH₂O—], 5.31 (d, 1H, H-1, J=3.7, H-1 Fuc), 2.48 [m, 4H, 2×(—CH₂ —CO)], 2.39 (m, 4H, COCH₂ CH₂CH₂CH₂ CO), 2.18 [m, 8H, 2×(—CH₂ —CH=CH—CH₂ —)], 1.93 (m, 2H, O—CH₂CH₂ CH₂—NH), 1.8 [m, 8H, COCH₂CH₂ CH₂ CH₂CO and 2×(COCH₂CH₂ —)]1.43, 1.47 (2 bs, 40H, 20 CH₂), 1.36 (d, 3H, J=6.6, CH3 Fuc), 1.05 (m, 6H, 2 CH₃).

H_(tri)-sp-Ad-DOPE (VII)—5.5 [m, 4H, 2×(—CH=CH—)], 5.4 (m, 1H, —OCH₂—CHO—CH₂O—), 5.35 (d, 1H, H-1, J=3.2, H-1 Fuc), 4.65, 4.54 (2d, J=7.4, J=8.6, H-1 Gal, H-1 GlcNHAc), 4.46 (dd, 1H J=3.18, J=12, —CCOOHCH—CHO—CH₂O—), 4.38-4.28 (m, 2H, H-5 Fuc, CCOOHCH—CHO—CH₂O—), 2.48 [m, 4H, 2×(—CH₂ —CO)], 2.40 (m, 4H, COCH₂ CH₂CH₂CH₂ CO), 2.18 [m, 8H, 2×(—CH₂ —CH—CH—CH₂—)], 2.08 (s, 3H,NHAc), 1.92 (m, 2H, O—CH₂CH₂ CH₂—NH), 1.82-1.72 [m, 8H, COCH₂CH₂ CH₂ CH₂CO and 2×(COCH₂CH₂ —) ], 1,48, 1.45 (2 bs, 40H, 20 CH₂), 1.39 (d, 3H, J=6.5, CH₃ Fuc), 1.05 (m, 6H, 2 CH₃) .

H_(di)-sp-Ad-DOPE (VIII)—5.49 (m, 4H, 2—(—CH=CH—), 5.37 (m, 1H, —OCH₂—CHO—CH₂O-13 ), 5.24 (d, 1H, H-1, J=2.95, H-1 Fuc), 4.46 (d, J=7.34, H-1 Gal), 2.48 [m, 4H, 2×(—CH₂ —CO)], 2.42-2.35 (m, 4H, COCH₂ CH₂CH₂CH₂ CO), 2.17 [m, 8H, 2≤(—CH₂ —CH=CH—CH₂ —)], 1.95 (m, 2H, O—CH₂CH₂ CH₂—NH), 1.81-1.74 [m, 8H, COCH₂CH₂ CH₂ CH₂CO and 2—(COCH₂CH₂ —)], 1.45, 1.41 (2 bs, 40H, 20 CH₂), 1.39 (d, 3H, J=6.5, CH₃ Fuc), 1.03 (m, 6H, 2 CH₃).

Galp-sp-Ad-DOPE (IX)—5.51 [m, 4H, 2×(—CH=CH—)], 5.4 (m, 1H, —OCH₂—CHO—CH₂O—), 4.61 (dd, 1H J=3.18, J=12, —CCOOHCH—CHO—CH₂O—), 4.41 (d, J=7.8, H-1 Gal), 4.37 (dd, 1H, J=6.6, J=12, —CCOOHCH—CHO—CH₂O—), 2.50 [m, 4H, 2×(—CH₂ —CO)], 2.40 (m, 4H, COCH₂ CH₂CH₂CH₂ CO), 2.20 [m, 8H, 2×(—CH₂ —CH=CH—CH₂ —)], 1.97 (m, 2H, O—CH₂CH₂ —CH₂—NH), 1.82-1.72 [m, 8H, COCH₂CH₂ CH₂ CH₂CO and 2—(COCH₂CH₂ —)], 1.48, 1.45 (2 bs, 40H, 20 CH2), 1.05 (m, 6H, 2 CH3).

Preparation of GalNAcα1-3Galβ1-4GlcNAc-Ad-DOPE (SCHEME VII)

To a solution of the product 8 (33 μmol) in N,N-dimethylformamide (1 ml), 30 μl mol of the 3-aminopropyltrisaccharide 5 and 5 μl of triethylamine (Et₃N) were added. The mixture was stirred for 2 h at room temperature. Column chromatography on silica gel (CH₂Cl ₂-EtOH—H₂O; 6:5:1) provided an 81% yield of the construct 9.

9: ¹H NMR (700 MHz, CDCl₃—CD₃OD, 1:1 v/v, selected), δ, ppm: 1.05 (t, 6H, J 7.05, 2 CH₃ ), 1.39-1.55 (m, 40H, 20 CH₂ ), 1.75-1.84 (m, 8H, COCH₂CH ₂CH ₂CH₂CO and 2×COCH₂CH ₂—) , 1.84-1.96 (m, 2H, O—CH₂CH ₂CH₂—NH), 2.15-2.22 (m, 14H, 2×(—CH ₂—CH=CH—CH ₂—), 2×NHC(O)CH ₃), 2.34-2.46 (m, 4H, 2×—CH₂-CO), 2.36-2.44 (m, 4H, 2×—CH ₂—CO), 3.29-3.34 (m, 1H, —CH₂—CHH—NH), 4.17-4.20 (m, 2H, —CHO—CH ₂OP—) 4.34-4.39 (m, 2H, —CH₂OPO—CH₂ —CH₂), 4.57 (d, 1H, J_(1,2) 8.39, H-1^(T)), 4.50 (dd, 1H, J 3.78, J 10.82, —C(O)OCHHCHOCH₂O—), 4.58-4.61 (m, 2H, H-1^(TT), C (O)OCHHCHOCH₂O—), 5.15 (d, 1H, J_(1,2) 3.76, H⁻1^(TTT)), 5.38-5.42 (m, 1H, —OCH₂—CHO—CH₂O—), 5.47-5.53 (m, 4H, 2×—CH=CH—). R_(f) 0.5 (CH₂Cl₂-EtOH-H₂O; 6:5:1).

Preparation of Gb₃-S₁ (sp3) -Ad-DOPE and Gb₃-S₁ (sp2) -Ad-DOPE (SCHEME VIII)

To a solution of activated DOPE (19) (10.5 μmol) in dichloromethane (300 μl) was added (12) or (16) (10 μmol) in DMF (0.5 ml) and then triethylamine (3 μl). The mixture was kept for 2 h at room temperature. Gel filtration on Sephadex LH-20 (1:1 (v/v) chloroform-methanol) of the mixture yielded (I) or (III) (90-95%).

Gb₃-sp3-Ad-DOPE (I) was determined to have a molecular weight (MW) of 1415.7 and ¹H NMR (CDCl₃/CD₃OD, 2:1), δ: 5.5 (m, 4H, 2×(—CH=CH—), 5.43-5.39 (m, 1H, —OCH₂—CHO—CH₂O—), 5.13 (d, 1H, J=3.6, H-1 Gal), 4.61-4.58 (m, 2H; J=7.1, H-1 (Gal); J=3.7, J=12.1, —CCOOHCH—CHO—CH₂O—), 4.46 (d, J=7.9, H-1 Gal), 2.53-2.48 (m, 4H, 2×(—CH2—CO), 2.42-2.37 (m, 4H, COCH ₂CH₂CH₂CH ₂CO), 2.21-2.16 (m, 8H, 2×(—CH ₂—CH=CH—CH ₂—), 2.00-1.95 (m, 2H, O—CH₂CH ₂CH₂—NH), 1.78 (m, 8H, COCH₂CH ₂CH ₂CH₂CO and 2×(COCH₂CH ₂—), 1.50, 1.47 (2 bs, 40H, 20 CH₂), 1.05 (m, 6H, 2 CH₃) (FIG. 1).

Gb₃-sp2-Ad-DOPE (III) was determined to have a molecular weight (MW) of 1415.7 and ¹H NMR (CDCl₃OD, 2:1), δ: 5.5 (m, 4H, 2×(—CH=CH—), 5.43-5.39 (m, 1H, —OCH₂—CHO—CH₂O—), 5.13 (d, 1H, J=3.6, H-1 Gal), 4.61-4.58 (m, 2H; J=7.1, H-1 (Gal); J=3.7, J=12.1, —CCOOHCH—CHO—CH₂O—), 4.46 (d, J=7.9, H-1 Gal), 2.53-2.48 (m, 4H, 2×(—CH ₂—CO), 2.42-2.37 (m, 4H, COCH ₂CH₂CH₂CH₂CO), 2.21-2.16 (m, 8H, 2×(—CH ₂—CH=CH—CH ₂—), 1.78 (m, 8H, COCH₂CH ₂CH ₂CH₂CO and 2×(COCH₂CH ₂—), 1.50, 1.47 (2 bs, 40H, 20 CH2), 1.05 (m, 6H, 2 CH3).

Preparation of Galili-sp-Ad-DOPE (SCHEME IX)

The reactions were performed with the use of commercial reagents (Acros, Aldrich, and Fluka); anhydrous solvents were purified according to the standard procedures. Column chromatography was performed on Silica gel 60 0.040-0.063 mm (Merck), gel filtration was carried out on Sephadex LH-20 (GE

Healthcare) columns. Solvents were removed in vacuum at 30-40 ° C. Thin layer chromatography (TLC) was performed on Silica gel 60 F254 aluminium-backed plates (Merck). Spots of compounds were visualized by dipping a TLC plate into aqueous solution of H₃PO₄ (8%) and subsequent heating (>150° C.).

¹H NMR spectra were recorded on a Bruker BioSpin GmbH (700 MHz) spectrometer at 30° C.; chemical shifts (δ, ppm) were referred to the peak of internal D₂O (δ 4.750), CDCl₃ (δ 7.270), or CD₃OD (δ 3.500); coupling constants (J) were measured in Hz. Signals of ¹H NMR. Symbols of monosaccharide residues in NMR spectra for saccharides: I—β-GlcNAc (reducing end), II—β-Gal, III—α-Gal. MALDI TOF MS spectra were recorded on Bruker Daltonics Ultraflex MALDI TOF/TOF Mass Spectrometer (Germany).

To a solution of 3-aminopropyl 4-O-[3-O-(α-D-galactopyranosyl)-β-D-galactopyranosyl]-2-acetamido-2-deoxy-β-D-glucopyranoside (33) (Mendeleev Communications, 2002, (143-145) or Tetrahedron, 61, (2005), 4313-4321), 52 mg, 0.086 mmol) in dry DMF (2 mL) was added 15 μL of Et₃N followed by a solution of DOPE-Ad-ONSu (31) (U.S. Pat. No. 8,013,131 B2, 100.6 mg, 1.00 mmol) in CH₂C₁₂ (2 mL). The reaction was stirred for 2 hours at room temperature followed by sequential column chromatography (the first on Sephadex LH-20, and the second on silica gel eluting with CH₂Cl₂-EtOH-H₂O; 6:5:1) to provide the construct designated Galili-sp-Ad-DOPE (34) (105.6 mg, 84%).

R_(f) 0.5 (CH₂Cl₂-EtOH-H₂O; 6:5:1). ¹H NMR (700 MHz, CDCl ₃—CD₃OD 1:1, 30° C.), δ ppm, selected: 5.45-5.54 (m, 4H, 2×—CH=CH—), 5.34-5.43 (m, 1H, —OCH₂—CHO—CH₂O—), 5.18 (d, 1H, J,,₂ 2.52, H-1^(TTT)), 4.61 (d, 1H, J_(1,2) 7.57, H-1^(TT)), 4.60 (dd, 1H, J 2.87, J 12.00, C(O)OCHHCHOCH₂O—), 4.56 (d, 1H, J_(1,2) 8.39, H-1^(T)), 4.36 (dd, 1H, J 6.8, J 12.00, —C(O)OCHHCHOCH₂O—), 4.19 (d, 1H, J_(3,4) 2.48, H-4^(TT)) , 4.13-4.18 (m, 2H, —CHO—CH ₂OP—), 3.52-3.62(m, 3H, PO—CH₂—CH ₂—NH, —CH₂—CHH—NH), 3.29-3.35 (m, 1H, —CH₂—CHH—NH), 2.45-2.52 (m, 4H, 2×—CH ₂—CO), 2.36-2.45 (m, 4H, 2×—CH₂—CO), 2.14-2.22 (m, 11H, 2×(—CH ₂—CH=CH—CH ₂—), NHC(O)CH ₃), 1.85-1.96 (m, 2H, O—CH₂CH ₂CH₂NH), 1.73-1.84 (m, 8H, COCH₂CH ₂CH ₂CH₂CO and 2×(COCH₂CH ₂—), 1.36 -1.55 (m, 40H, 20 CH ₂), 1.05 (t, 6H, J 6.98, 2 CH ₃). C₇₀H₁₂₆N₃O₂₆P; MALDI MS: m/z 1480 (M Na+H); 1496 (MK+H); 1502 (MNa+Na), 1518 (M Na+K)

Condensation of DOPE-A with 5-((5-aminopentyl)thioureidyl) Fluorescein (Fluorescein Cadaverine)

To a solution of activated DOPE (L-A) (5 mg, 5.2 μmol) in N,N-dimethylformamide (0.5 ml) 3 mg (4.6 μmol) of fluorescein cadaverine dihydrobromide salt and 5 μl of triethylamine were added. The mixture was kept for 2 h at room temperature, then 10 μl of 3% aq. NH₃ were added and the mixture was kept at room temperature for 1 h.

Column chromatography (Sephadex LH-20, 1:1 chloroform-methanol, followed by silica gel, ethyl acetate-isopropanol-water, 6:3:1) of the mixture yielded 4.2 mg (67%) KODE-fluorescein (I), R_(f) 0.5 (ethyl acetate-isopropanol-water, 6:3:1).

¹H NMR (CDCl₃/CD₃OD, 1:1), δ:

KODE-fluorescein (I)—8.38 (bs, 1H, aromatic proton of fluorescein), 8.15 (dd, 1H, J=1.7, J=8.3, aromatic proton of fluorescein) 7.30(d, 1H, J=8.3, aromatic proton of fluorescein), 6.87 (m, 4H, aromatic protons of fluorescein), 6.72 (dd, 2H, J=2.4, J=8.8, aromatic protons of fluorescein), 5.50 (m, 4H, 2×(—CH=CH—), 5,38 (m, 1H, —OCH₂-CHO—CH₂O—), 4.58 (dd, 1H, J=6.6, J_(gem)=11.8, HHC—O—C(O)—), 4.34 (dd, 1H, J=3.2, J_(gem)=11.8, HHC—O—C(O)—), 4.14 (m, 2H, —OCH—CH₂ —O—P—) (4.1 (m, 2H, —P—O—CH₂ —CH₂—NH—) 3.80 (m, 2H, N—CH₂(CH₂)₃—CH₂ NH—C=S) 3.39 and 3.58 (2 m, 2×2H, N— CH₂—CH₂—O—P— and N—CH₂—(CH₂)₃—CH₂NH—C=S)2.48 (m, 4H, 2×(—CH₂ —CO), 2.39 (m, 4H, COCH₂ CH₂CH₂CH₂ CO), 2.19 (m, 8H, 2×(—CH₂ —CH=CH —CH₂ —), 1.84 (m, 2H, CH₂ - fluorescein cadaverine), 1.8 (m, 10H, COCH₂CH₂ CH₂ CH₂CO, 2×(COCH₂CH₂ —, and CH₂ - fluorescein cadaverine), 1.62 (m, 2H, CH₂ — fluorescein cadaverine) 1.42, 1.46 (2 bs, 40H, 20 CH2), 1.05 (m, 6H, 2 CH₃).

Condensation of DOPE-A with 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl ethylenediamine, hydrochloride (BODIPY FL EDA)

To a solution of 15 mg (15.5 pmol) activated DOPE in CH₂Cl₂ (0.5 ml), 5 mg (13.5 pmol) of BODIPY FL EDA in N,N-dimethylformamide (0.3 ml) and 5 μl of triethylamine were added. The mixture was kept for 2 h at room temperature.

Column chromatography (Sephadex LH-20, 1:1 chloroform-methanol) of the mixture yielded 14.2 mg (75%) KODE-BODIPY (I), Et₃N-salt; MW 1289.6, R_(f) 0.3 (ethyl acetate-isopropanol-water, 6:3:1).

¹H NMR (CDCl₃/CD₃OD, 1:1): δ 7.40 (s, 1H, aromatic proton of BODIPY), 7.12 (d, 1H, J=3.8 aromatic proton of BODIPY), 6.47 (d, 1H, J=3.8 aromatic proton of BODIPY), 6.32 (s, 1H, aromatic protons of BODIPY), 5.50 (m, 4H, 2x(—CH=CH—), 5.38 (m, 1H, —OCH₂—CHO—CH₂O—), 4.58 (dd, 1H, J3.2, J_(gem)=11.8, HHC—O—C(O)—), 4.33 (dd, 1H, J=6.6, J_(gem)=11.8, HHC—O—C(O)—), 4.16 (t, 2H, J=5.6, P—O—CH ₂—CH₂—NH—), 4.1 (m, 2H, — —OCH—CH ₂—O—P—), 3.60 (t, 2H, P—O—CH₂—CH ₂—NH—), 3.46, 3.42 and 2.8 (3m, 4H, 2H, 2H, —CH ₂—CH ₂—C(O)NH(CH ₂)₂—NH of BODIPY), 2.70 (s, 3H, CH₃ of BODIPY), 2.48 (m, 4H, 2x (—CH₂—CO) , 2.45 (s, 3H, CH₃ of BODIPY), 2.37 (m, 4H, COCH ₂CH₂CH₂CH ₂CO), 2.19 (m, 8H, 2x(—CH ₂—CH=CH—CH ₂—), 1.8 (m, 8H, COCH₂CH₂CH ₂CH ₂CO, 2×(COCH₂CH₂-)), 1.46, 1.43 (2 bs, 40H, 20 CH₂), 1.05 (m, 6H, 2 CH₃); 3.31 (q, 6H, J=7.4, 3×CH₂ of Et₃N), 1.50 (t, 9H, J=7.4, 3×CH₃ of Et₃N).

Example 2 Solubility of Synthetic Glycolipids

For use in the transformation of cells the first criterion that synthetic glycolipids must satisfy is that they are soluble in aqueous solvents, e.g. phosphate buffered saline. A number of techniques, including heat and/or sonication, were employed initially in order to maximise the solubility of the synthetic glycolipids tested (Table 21).

The synthetic glycolipid must also be able to insert into the membrane and be recognisable to the appropriate antibody for transformation to be detected by agglutination. Initial tests on the molecules were to establish solubility and thus eliminate those molecules that were unsuitable for use in the transformation of cells.

The results of these initial tests are provided in Table 22.

TABLE 21 The range of synthetic glycolipid molecules tested. DOPE Lipid Tails: B_(tri)-sp-Ad-DOPE (VI) A_(tri)-sp-Ad-DOPE (I) Galβ-sp-Ad-DOPE (IX) H_(di)-sp-Ad-DOPE (VIII) H_(tri)-sp-Ad-DOPE (VII) A_(tri)-spsp₁-Ad-DOPE (II) B_(tri)-PAA-DOPE (V) Different Lipid Tails: A_(tri)-sp-lipid (IV) A_(tri)-sp-Ad-DSPE (III)

TABLE 22 Solubility of synthetic glycolipids in hot PBS and transformation ability. Water Detectable Synthetic solubility transformation ability A_(tri)-sp-lipid (IV) No No B_(tri)-PAA-DOPE (V) No No B_(tri)-sp-Ad-DOPE (VI) Yes Yes A_(tri)-sp-Ad-DOPE (I) Yes Yes Galβ-sp-Ad-DOPE Yes No (IX) H_(di)-sp-Ad-DOPE Yes No (VIII) H_(tri)-sp-Ad-DOPE Yes Yes (VII) A_(tri)-spsp₁-Ad-DOPE Yes Yes (II) A_(tri)-sp-Ad-DSPE Yes Yes (III)

The lack of detectable transformation for Gaβ-sp-Ad-DOPE (IX) and H_(di)-sp-Ad-DOPE (VIII) was thought to be due to the inability of the antibody to recognise the glycotope of these synthetic molecules. A_(tri)-sp-lipid (IV) has a single rather than a diacyl tail and it was proposed that there was no insertion of this synthetic molecule into the membrane bilayer.

Example 3 Low Temperature Transformation of RBCS by A_(tri)-sp-Ad-DOPE (I) and B_(tri)-sp-Ad-DOPE (VI) Synthetic Glycolipids

RBCs are healthier when stored at 4° C., and likewise are believed to be healthier when transformed at 4° C. It was not thought that a significant rate of insertion of the synthetic glycolipids would occur at 4° C. due to our previous studies (see Comparative Examples) and studies by others

(Schwarzmann, 2000). These studies were performed with natural glycolipids. Surprisingly these studies did not predict the behaviour of the synthetic glycolipids of the invention.

Whilst not wishing to be bound by theory, in the studies of Schwarzmann the low rate of insertion of the natural glycolipids may be due to the physicochemical properties of the natural glycolipid tail; a sphingolipid and a fatty acid.

The diacyl tail of the glycolipid may be important in determining the rate of insertion. Certain diacyl tails may retain greater fluidity at lower temperatures. Alternatively, the domain of the plasma membrane into which the diacyl tail of these glycolipids inserts may retain this greater fluidity.

It is known that the sphingolipid tails of natural glycolipids congregate in rigid domains and these domains may not allow further incorporation of glycolipid at low temperatures. Synthetic glycolipids with cis-desaturated diacyl tails may be favoured for use.

Transformation of RBCs with synthetic glycolipids with different lipid tails was first evaluated (Tables 22 and 24).

TABLE 23 Antisera used to obtain results presented in Tables 24 to 27. Manufacturer Catalogue ref Batch number Expiry date Anti-A Albaclone, SNBTS Experimental Z0010770 12.12.04 BioClone, OCD reagent 01102 — Anti-B Albaclone, SNBTS Experimental Z0110600 27.04.03 BioClone, OCD reagent 01103 —

Transformation of RBCs with synthetic glycolipids A_(tri)-sp-Ad-DOPE (I) and B_(tri)-sp-Ad-DOPE (VI) at 4° C. was then evaluated (Tables 25 to 28). These transformations were directed towards the preparation of cells expressing low levels of A, B or A and B glycotopes (“weak A, B and AB cells”).

For the preparation of weak A and B cells transformation solutions (20 μl, A_(tri)-sp-Ad-DOPE (I) at 0.08, 0.05 and 0.03 mg/mL, and B_(tri)-sp-Ad-DOPE (VI) at 0.6, 0.3, 0.15, 0.08, 0.05 and 0.03 mg/mL) in 1×PBS were mixed with washed, packed group O RBCs (60 μL).

For the preparation of weak AB cells transformation solutions (20 μL, A_(tri)-sp-Ad-DOPE (I) at 0.07, 0.06 and 0.05 mg/mL, and B_(tri)-sp-Ad-DOPE (VI) at 0.3, and 0.2 mg/mL) in 1×PBS were combined in block titre with washed, packed group O RBCs (60 μL). The combinations were: A_(tri)-sp-Ad-DOPE (I) at 0.07 mg/mL+B_(tri)-sp-Ad-DOPE (VI) at 0.3 mg/mL; A_(tri)-sp-Ad-DOPE (I) at 0.07 mg/mL,+B_(tri)-sp-Ad-DOPE (VI) at 0.2 mg/mL; A_(tri)-sp-Ad-DOPE (I) at 0.06 mg/mL+B_(tri)-sp-Ad-DOPE (VI) at 0.3 mg/mL; A_(tri)-sp-Ad-DOPE (I) at 0.06 mg/mL+B_(tri)-sp-Ad-DOPE (VI) at 0.2 mg/mL; A_(tri)-sp-Ad-DOPE (I) at 0.05 mg/mL+B_(tri)-sp-Ad-DOPE (VI) at 0.3 mg/mL; and A_(tri)-sp-Ad-DOPE (I) 0.05+B_(tri)-sp-Ad-DOPE (VI) 0.2 mg/mL.

TABLE 24 Evaluation of insertion of different lipid tails by agglutination with the relevant antisera. Anti- Transformation solution (μg/mL) Molecule sera 1000 500 250 125 100 60 50 40 30 20 10 A_(tri)-sp-Ad- Alba w+ w+ 0 0 0  DOPE (I) Bio 2+  1+ w+ 0 0  Alba 4+ 3+ 2-3+ 2+ Bio  4+*  4+*  3+* 3+ DBA 0 B_(tri)-sp-Ad- Alba  3+ DOPE (VI) Bio  3+ Alba  2+ 2+ 1+ 0 0 Bio  3+ 2+ 1+ 0 0 A_(tri)-spsp₁-Ad- Alba 0 0 0 0 0  DOPE (III) Bio 0 0 0 0 0  Alba 4+ 3+ 2+ 2+ Bio  4+*  3-4+*  3+* 2+ DBA 0 A_(tri)-sp-lipid Alba 0 (IV) Bio 0 A_(tri)-sp-Ad- Alba 0 0 0 0 0  DSPE (III) Bio 0 0 0 0 0  Alba 2-3+ 2-3+ 2+ 2+ Bio 3+ 2-3+ 2+ 2+ DBA 0 *splatter.

Cells and transformation solutions were placed in a 4° C. fridge. Pipette mixing was performed at intervals. Cells were removed for testing at intervals against the relevant antisera and were tested in both washed and unwashed states (i.e. washed samples had the transformation solution removed).

After 48 hours Celpresol™ was added to the cells so that the final cells:non-cells ratio was 3:5 (v/v). The cells continued to be tested at intervals. Testing was discontinued after 10 days because cells turned brown.

This discolouration could be attributed to a number of factors including: cells were already 21 days old when transformed; 48 hour transformation was in PBS not Celpresol™ so cells stressed for this time; and cells may have been mishandled in transit between the transforming and testing laboratories. This may be mitigated by transformation of the cells in Celpresol™ as opposed to PBS.

TABLE 25 Diamed results of weak A RBCs transformed at 4° C. against anti-A. A_(tri)-sp-Ad-DOPE (I) (mg/mL) Washed unwashed Time 0.08 0.05 0.03 0.08 0.05 0.03 2 hrs 0  0 0 0  0  0 4 hrs 1+ 0 0 2+ 0  0 6 hrs 2+ 0 0 2+ 0  0 8 hrs 2+ 0 0 2-3+ 0  0 12 hrs 2-3+ 0 0 3+ 1+ 0 24 hrs 3-4+  1+ 0 3-4+ 2+ 0 30.5 hr 3-4+  1+ 0 3-4+ 2+ 0 48 hrs 4+  2+ 0 4+ 2+ 0 72 hrs 4+  2+ 0 4+ 2-3+ 0 96 hrs 4+ 2-3+ 0 4+ 2-3+ 0 Day 7 3-4+ 2+ 0 Day 10 3-4+ 2+ 0

TABLE 26 Diamed results of weak B RBCs transformed at 4° C. against anti-B. B_(tri)-sp-Ad-DOPE (VI) (mg/mL) washed unwashed Time 0.6 0.3 0.15 0.6 0.3 0.15 2 hrs 0  0 0 0  0  0 4 hrs 0  0 0 1+ 0  0 6 hrs w+ 0 0 1+ 0  0 8 hrs 2+ 0 0 2+ w+ 0 12 hrs 2+  w+ 0 2-3+ 2+ 0 24 hrs 4+  3+  2+ 4+ 3+  2+ 30.5 hr 4+ 2-3+ 0 4+ 2-3+  w+ 48 hrs 4+  3+  1+ 4+ 3+  2+ 72 hrs 4+  4+  2+ 4+ 4+  2+ 96 hrs 4+ 3-4+ 2-3+ 4+ 3-4+ 2-3+ Day 7 4+ 2-3+ 0 Day 10 4+ 2+ 0

TABLE 27 Diamed results of weak AB RBCs transformed at 4° C. in block titre against anti-A. B_(tri)-sp-Ad- A_(tri)-sp-Ad-DOPE (I) (mg/mL) DOPE (VI) washed unwashed Day (mg/mL) 0.07 0.06 0.05 0.07 0.06 0.05 1 0.3 2+ 1-2+ w+ 2-3+ 2+ 1+ 0.2 2+ 1-2+ 0  2-3+ 2+ 1+ 5 0.3 2+ 1-2+ 1+ 2-3+ 2+ 1-2+ 0.2 2+ 1-2+ w+ 2-3+ 2+ 1-2+ 8 0.3 2-3+ 2+ 2+ 0.2 2-3+ 2+ 1-2+

Example 4 Insertion Efficiency of Transformation of RBCS by A_(tri)-sp-Ad-DOPE

(I) and B_(tri)-sp-Ad-DOPE (VI) Synthetic Glycolipids

The post-transformation supernatant solutions (from A_(tri)-sp-Ad-DOPE (I) at 0.08 mg/mL, 0.05 mg/mL and 0.03 mg/mL, and B_(tri)-sp-Ad-DOPE (VI) at 0.6 mg/mL, 20 μL) were added neat and in a 1:2 dilution to washed, packed RBCs (60 μL). The tubes were incubated in a 37° C. waterbath for one hour, with mixing taking place every 15 minutes.

TABLE 28 Diamed results of weak AB RBCs transformed at 4° C. in block titre against anti-B. B_(tri)-sp-Ad- A_(tri)-sp-Ad-DOPE (I) (mg/mL) DOPE (VI) washed unwashed Day (mg/mL) 0.07 0.06 0.05 0.07 0.06 0.05 1 0.3 3+ 3+ 2+ 3+ 3+ 2-3+ 0.2 1+ 1-2+ 0  2+ 2+ 1-2+ 5 0.3 2+ 2+ 1+ 2+ 2+ 2+ 0.2 0  w+ vw 1+ w+ vw 8 0.3 2+ 2+ 2+ 0.2 1+ 1+ 0 

The transformed RBCs were washed 3× with PBS and then suspended in Cellstab™ at the appropriate concentration for serology testing.

TABLE 29 Tube serology. Pre-trans conc (mg/mL) Score A_(tri)-sp-Ad-DOPE (I) at 0.08 0 1:2 of A_(tri)-sp-Ad-DOPE (I) at 0.08 0 A_(tri)-sp-Ad-DOPE (I) at 0.05 0 1:2 of A_(tri)-sp-Ad-DOPE (I) at 0.05 0 A_(tri)-sp-Ad-DOPE (I) at 0.03 0 1:2 of A_(tri)-sp-Ad-DOPE (I) at 0.03 0 B_(tri)-sp-Ad-DOPE (VI) at 0.60 vw+ 1:2 of B_(tri)-sp-Ad-DOPE (VI) at 0.60 0

The score given by the post-transformation supernatant solution (from the 0.08 mg/mL pre-transformation solution) is not even that of the 0.03 mg/mL transformation solution in the first pass (w+). These results indicate that >75% of the molecules are inserted into the RBC membrane on the first pass.

In addition, the post-transformation solutions were concentrated 20× and compared in parallel with the transformation solutions of known concentration. Only the post-transformation solutions derived from the 0.08 mg/mL A_(tri)-sp-Ad-DOPE (I) and 0.6 mg/mL B_(tri)-sp-Ad-DOPE (VI) solutions were tested.

Post-transformation solutions (20 μL) were dialysed (pore size 500 Da) against de-ionised water for 2 days. The samples were left to dry in a fumehood for 10 days. At the end of this time they were transferred into a rotavapor flask and set on the rotavapor to rotate under vacuum with no heat overnight.

Samples were dried in a water bath at 40° C. and washed over into smaller vessels with chloroform-methanol 2:1 leaving significant amounts of dried cellular material. The chloroform-methanol 2:1 washings were dried down, washed over again into test-tubes with chloroform-methanol 2:1 and dried down. These samples were redissolved in 1 mL of 1×PBS and used for transformation experiments. The cellular material in the bottom of the flasks was washed out with water into another set of tubes.

The post-transformation solutions (from A_(tri)-sp-Ad-DOPE (I) at 0.08 mg/mL and B_(tri)-sp-Ad-DOPE (VI) at 0.6 mg/mL, 20 μL) were added to washed, packed RBCs (60 μL). In parallel, the transformation solutions (A_(tri)-sp-Ad-DOPE (I) at 0.08 mg/mL, 0.05 mg/mL and 0.03 mg/mL, and B_(tri)-sp-Ad-DOPE (VI) at 0.6 mg/mL, 20 μL) were added to washed, packed RBCs (60 μL).

The tubes were incubated in a 37° C. waterbath for one hour, with mixing taking place every 15 minutes. The transformed RBCs were washed 3x with PBS and then suspended in Cellstab™ at the appropriate concentration for serology testing.

TABLE 30 Diamed serology. Conc (mg/mL) Score A_(tri)-sp-Ad-DOPE (I) at 0.08 3+ A_(tri)-sp-Ad-DOPE (I) at 0.05 2+ A_(tri)-sp-Ad-DOPE (I) at 0.03 1+ From A_(tri)-sp-Ad-DOPE (I) at 0.08 0 B_(tri)-sp-Ad-DOPE (VI) at 0.60 4+ From B_(tri)-sp-Ad-DOPE (VI) at 0.60 0

These results suggest that there are not enough molecules in the post-transformation solution, even when concentrated 20×, to be detected by serology.

Example 5 Transformation of Murine RBCS by H_(tri)-sp-Ad-DOPE (VII) Synthetic Glycolipid

Mouse cells were transformed at 37° C. for 1 hour.

TABLE 31 Anti-H reagents used for results in Tables 32 and 33. Antisera Manufacturer Batch Anti-H IgM Japanese Red Cross HIRO-75 UEA Lorne Laboratories 11549E D.O.E. 06.2004 Bio-UEA EY Labs 201105-2

TABLE 32 Tube Serology. H Antisera UEA Cells IgM T = 0 T = 20 Bio-UEA Mouse RBCs (- control) 0 0 0 Mouse RBCs + 0.01 mg/mL 0 H_(tri)-sp-Ad-DOPE (VII) Mouse RBCs + 0.05 mg/mL 1+ H_(tri)-sp-Ad-DOPE (VII) Mouse RBCs + 0.1 mg/mL 3+ H_(tri)-sp-Ad-DOPE (VII) Mouse RBCs + 0.25 mg/mL 4+ 1+ H_(tri)-sp-Ad-DOPE (VII) Mouse RBCs + 1 mg/mL 2+ 2+ H_(tri)-sp-Ad-DOPE (VII) Human O RBCs (+ control) 4+ 1+ 2/3+ 4+

TABLE 33 Diamed. Cells Score Mouse RBCs + 0.01 mg/ml H_(tri)-sp-Ad-DOPE (VII) 0 Mouse RBCs + 0.05 mg/ml H_(tri)-sp-Ad-DOPE (VII) 0 Mouse RBCs + 0.1 mg/ml H_(tri)-sp-Ad-DOPE (VII) 2+ Mouse RBCs + 0.25 mg/ml H_(tri)-sp-Ad-DOPE (VII) 3+

Example 6 Transformation of RBCs by Filtered A_(tri)-sp-Ad-DOPE (I) Synthetic Glycolipid

Some A_(tri)-sp-Ad-DOPE (I) had been sterile-filtered through a 0.2 μm filter. To investigate whether transformation would be the same with this product a comparative trial was done.

TABLE 34 Anti-A used for results presented in Table 35. Manufacturer Catalogue ref Batch number Expiry date BioClone, OCD Experimental 01102 — reagent

TABLE 35 Column agglutination of A RBCs transformed with varying concentrations of sterile-filtered vs unfiltered A_(tri)-sp-Ad-DOPE (I). Concentration Sterile-filtered A_(tri)-sp-Ad- Unfiltered A_(tri)-sp-Ad- (mg/mL) DOPE (I) DOPE (I) 0.2 4+ 4+ 0.1 4+ 3-4+ 0.05 2-3+ 2-3+ 0.01 0 0 Control 37° C. 0 Control 25° C. 0

These results show no significant difference between the two preparations of A_(tri)-sp-Ad-DOPE (I) and suggests that filtration through a 0.2 μM filter did not remove molecules or change the composition or properties of the fluid to the point that transformation was affected.

Example 7 Storage of Transformed Cells

To investigate whether storage at 4° C. or 37° C. changed the agglutination results of A_(tri)-sp-Ad-DOPE (I) and natural A glycolipid transformed O RBCs, identified as “Syn-A” and “Nat-A” cells respectively, were divided in two and suspended to 5% in Cellstab™.

One set of cells was stored at 4° C. and the other set of cells was stored at

37° C. in a waterbath. Agglutination of the stored transformed cells was assessed (Table 36).

TABLE 36 Syn-A A_(tri)-sp-Ad- Nat-A Time Plat- Temp DOPE (I) at At At (hours) form (° C.) 0.1 mg/mL 1 mg/mL 10 mg/mL Control 0 Tube 3+ 0 1-2+ 0 20 Column 4 4+ 0 3+ 0 37 4+ 0 3+ 0 44 Column 4 4+ 3+ 0 37 4+ 3+ 0

Example 8 RBC Transformation with A- and B-antigen Synthetic Glycolipids with Different Non-Carbohydrate Structures

The water soluble synthetic glycolipids designated A_(tri)-sp-Ad-DOPE (I), A_(tri)-sp₁sp₂-Ad-DOPE (II), A_(tri)-sp-Ad-DSPE (III), and B_(tri)-sp-Ad-DOPE (VI) were prepared according to the method described in Example 1 with necessary modifications.

Washed packed group O red blood cells (RBCs) (3 parts by volume) and the synthetic glycolipid solution (1 part by volume, varying concentrations) were added to an eppendorf tube. The tube was incubated in a 37° C. waterbath for one hour, mixing every 15 minutes. The transformed RBCs were washed 3× with PBS and then suspended in Cellstab™ at the appropriate concentration for serology testing.

Tube serology and Diamed gel-card results for RBCs transformed with the different synthetic molecule constructs are provided in Table 38. Results for the stability of the RBCs transformed with the different synthetic glycolipids at different concentrations are provided in Tables 39 to 44.

TABLE 37 Antisera used for results presented in Tables 38 to 44. Antisera Manufacturer Batch Albaclone anti-A SNBTS Z0010770 - D.O.E 12.12.04 Bioclone anti-A Ortho Diagnostics 01102 - D.O.M 16.05.02 Albaclone anti-B SNBTS Z0110670 - D.O.E 12.12.04 Bioclone anti-B Ortho Diagnostics 01103 - D.O.M 16.05.02

TABLE 38 Comparison of transformation of RBCs using A-antigen synthetic glycolipids at different concentrations. A Antisera Conc Albaclone anti-A Bioclone anti-A Synthetic mg/mL Tube Diamed Tube Diamed A_(tri)-sp-Ad-DOPE (I) 0.25 n.d. 4+ n.d. 4+ 0.1 n.d. 4+/3+ n.d. 4+/3+ 0.05 w+ 2+ 2+ 2+ 0.04 w+ n.d. 1+ n.d. 0.03 0 n.d. w+ n.d. 0.02 0 n.d. 0 n.d. 0.01 0 0 0 0 A_(tri)-sp-Ad-DSPE (III) 0.25 n.d. 0 n.d. 0 0.1 n.d. 0 n.d. 0 0.05 0 0 0 0 0.04 0 n.d. 0 n.d. 0.03 0 n.d. 0 n.d. 0.02 0 n.d. 0 n.d. 0.01 0 0 0 0 A_(tri)-sp₁sp₂-Ad-DOPE (II) 0.25 n.d. 4+ n.d. 4+ 0.1 n.d. 4+ n.d. 4+/3+ 0.05 0 3+ 0 3+ 0.04 0 n.d. 0 n.d. 0.03 0 n.d. 0 n.d. 0.02 0 n.d. 0 n.d. 0.01 0 0 0 0 Incubated control — 0 n.d. 0 n.d. Bench control — 0 n.d. 0 n.d. Abbreviations: n.d. Not determined.

TABLE 39 Stability trial of RBCs transformed with Atri-sp-Ad- DOPE (I) at high concentrations (1 mg/mL, 0.5 mg/mL and 0.25 mg/mL). Agglutination by manual tube serology. Cell Albaclone anti-A Bioclone anti-A storage Concentration of Transformation Solution (mg/mL) Day solution 1 0.5 0.25 1 0.5 0.25 2 Alsevers 4+ 4+ 4+ 4+^(o) 4+^(o) 4+^(o) Cellstab ™ 4+ 4+ 3+ 4+^(o) 4+^(o) 4+^(o) 10 Alsevers 3+ 2+ 2+ 4+^(o) 4+^(o) 3+  Cellstab ™  4+^(o)  3+^(o) 2+ 4+^(o) 4+^(o) 4+^(o) 17 Alsevers 4+ 4+ 4+ 4+^(o) 4+^(o) 4+^(o) Cellstab ™ 4+ 4+ 4+ 4+^(o) 4+^(o) 4+^(o) 24 Alsevers 4+ 4+ 4+ 4+  4+  4+  Cellstab ™ 4+ 4+ 4+ 4+^(o) 4+  4+ 

TABLE 40 Stability trial of RBCs transformed with A_(tri)-sp-Ad-DOPE (I) at low concentrations (0.1 mg/mL, 0.05 mg/mL and 0.025 mg/mL). Agglutination by manual tube serology. Cell Albaclone anti-A Bioclone anti-A storage Concentration of Transformation Solution (mg/mL) Day solution 0.1 0.05 0.025 0.1 0.05 0.025 2 Alsevers 3+/2+ 1+ 1+/w+ 2+ 2+/1+ 1+ Cellstab ™ 3+/2+ 2+  1+ 3+/2+ 3+/2+ 2+ 8 Alsevers 2+ 1+  w+ 3+/2+ 2+ 2+ Cellstab ™ 2+ 1+/w+ vw  3+^(o) 2+ 1+ 15 Alsevers 2+ 1+ 0 3+ 2+ Vw Cellstab ™ 4+ w+ 0 4+ 4+ 1+ 22 Alsevers 2+ 2+ 0 3+ 2+ w+ Cellstab ™ 4+ 4+  1+ 4+ 4+ 1+ 44 Alsevers n.d. n.d. n.d. n.d. n.d. n.d. Cellstab ™ 4+ 2+  w+ 4+ 2+ w+

TABLE 41 Stability trial of RBCs transformed with A_(tri)-sp-Ad-DOPE (I) at high concentrations (1 mg/mL, 0.5 mg/mL and 0.25 mg/mL). Agglutination in Diamed gel-cards. Cell Albaclone anti-A Bioclone anti-A storage Concentration of Transformation Solution (mg/mL) Day solution 1 0.5 0.25 1 0.5 0.25 2 Alsevers 4+ 4+ 4+ 4+ 4+ 4+ Cellstab ™ 4+ 4+ 4+ 4+ 4+ 4+ 10 Alsevers 4+ 4+ 4+ 4+ 4+ 4+ Cellstab ™ 4+ 4+ 4+ 4+ 4+ 4+ 17 Alsevers 4+ 4+ 4+ 4+ 4+ 4+ Cellstab ™ 4+ 4+ 4+ 4+ 4+ 4+ 24 Alsevers 4+ 4+ 4+ 4+ 4+ 4+ Cellstab ™ 4+ 4+ 4+ 4+ 4+ 4+ 45 Alsevers 4+ 4+ 4+ 4+ 4+ 4+ Cellstab ™ 4+ 4+ 4+ 4+ 4+ 4+ 59 Alsevers 4+ 4+ 4+ 4+ Cellstab ™ 4+ 4+ 4+ 4+ 4+ 4+ 73 Alsevers Cellstab ™ 4+ 4+ 4+ 4+ 4+ 4+ 88 Alsevers Cellstab ™ 4+ 4+ 4+ 4+ 4+ 4+

TABLE 42 Stability trial of RBCs transformed with A_(tri)-sp-Ad-DOPE (I) at low concentrations (0.1 mg/mL, 0.05 mg/mL and 0.025 mg/mL). Agglutination in Diamed gel-cards. Where there were insufficient cells for testing, blank spaces have been left. Cell Albaclone anti-A Bioclone anti-A storage Concentration of Transformation Solution (mg/mL) Day solution 0.1 0.05 0.025 0.1 0.05 0.025 2 Alsevers 4+ 2+ 0 4+ 3+ 1+ Cellstab ™ 4+ 2+ 0 4+ 3+ 1+ 8 Alsevers 4+ 3+ 0 4+ 4+ 1+ Cellstab ™ 4+ 3+ 0 4+ 4+ 1+ 15 Alsevers 4+ 2+ 0 4+ 3+/2+ 1+ Cellstab ™ 4+ 4+ 0 4+ 4+ 1+ 22 Alsevers 4+ 3+/2+ 0 4+ 3+ w+ Cellstab ™ 4+ 4+ 0 4+ 4+ 1+ 29 Alsevers 4+ 2+ 0 4+ 3+ w+ Cellstab ™ 4+ 3+ 0 4+ 4+ 2+ 43 Alsevers 4+ 3+  w+ 4+ 4+ 2+ Cellstab ™ 4+ 4+/3+ 0 4+ 4+ 1+ 50 Alsevers 4+ 3+  w+ 4+ 4+ 2+ Cellstab ™ 4+ 3+ 0 4+ 4+ 1+ 57 Alsevers 4+ 3+/2+ 4+ 4+ Cellstab ™ 4+ 3+ 0 4+ 3+ w+ 63 Alsevers Cellstab ™ 4+/3+ 2+ 0 4+ 3+ 0  71 Alsevers Cellstab ™ 4+/3+ 2+ 0 4+ 3+ 0  86 Alsevers Cellstab ™ 4+/3+ 2+ 0 4+ 3+ 0 

TABLE 43 Stability trial of RBCs transformed with B_(tri)-sp-Ad-DOPE (VI) at high concentrations (1 mg/mL, 0.5 mg/mL and 0.25 mg/mL). Agglutination by manual tube serology (^(o)—splatter). Cell Albaclone anti-B Bioclone anti-B storage Concentration of Transformation Solution (mg/mL) Day solution 1 0.5 0.25 1 0.5 0.25 2 Alsevers 3+ 3+ 2+ 2+ 1+ 1+ Cellstab ™ 3+ 2+ 2+ 2+ 2+ 1+ 9 Alsevers 4+ 4+ 2+ 4+ 3+ 2+ Cellstab ™ 4+ 4+ 3+ 4+ 4+ 2+ 16 Alsevers 4+ 4+ 3+ 4+ 4+ 2+ Cellstab ™ 4+ 4+ 2+ 4+ 4+ 2+ 23 Alsevers 4+ 4+ 3+ 4+ 4+ 3+ Cellstab ™ 4+ 4+ 3+ 4+ 4+ 3+ 30 Alsevers 3+ 3+ 2+ 2+ 2+ 2+ Cellstab ™ 4+ 3+ 2+  3+^(o)  3+^(o) 2+ 37 Alsevers 3+ 2+ 1+ 3+ 2+ 1+ Cellstab ™ 3+ 3+ 2+/1+  4+^(o) 3+ 1+ 44 Alsevers 4+ 3+ 1+ 3+ 3+ w+ Cellstab ™ 4+ 4+ n.d. 4+ 4+ ^(‡) 51 Alsevers 3+ 3+ 2+ 4+ 3+ 2+ Cellstab ™ 4+ 4+ n.d. 4+ 4+ 2+

TABLE 44 Stability trial of RBCs transformed with B_(tri)-sp-Ad-DOPE (VI) at high concentrations (1 mg/mL, 0.5 mg/mL and 0.25 mg/mL). Agglutination in Diamed gel-cards. Where there were insufficient cells for testing, blank spaces have been left. Cell Albaclone anti-B Bioclone anti-B storage Concentration of Transformation Solution (mg/mL) Day solution 1 0.5 0.25 1 0.5 0.25 2 Alsevers 4+ 4+ 2+ 4+ 4+ 2+ Cellstab ™ 4+ 4+ 2+ 4+ 4+ 2+ 9 Alsevers 4+ 4+ 2+ 4+ 4+ 2+ Cellstab ™ 4+ 4+ 3+ 4+ 4+ 3+ 16 Alsevers 4+ 4+ 2+ 4+ 4+ 1+ Cellstab ™ 4+ 4+ 3+ 4+ 4+ 3+ 23 Alsevers 4+ 4+ 3+ 4+ 4+ 3+ Cellstab ™ 4+ 4+ 3+ 4+ 4+ 3+ 30 Alsevers 4+ 4+ 3+ 4+ 4+ 3+ Cellstab ™ 4+ 4+ 3+ 4+ 4+ 3+ 37 Alsevers 4+ 4+ 3+ 4+ 4+ 3+ Cellstab ™ 4+ 4+ 3+ 4+ 4+ 3+ 44 Alsevers 4+ 4+ 2+ 4+ 4+ 3+ Cellstab ™ 4+ 4+ 3+ 4+ 4+ 4+/3+ 51 Alsevers 4+ 4+ 2+ 4+ 4+ 3+ Cellstab ™ 4+ 4+ 3+ 4+ 4+ 3+ 58 Alsevers 4+ 1+ 4+ 2+ Cellstab ™ 4+ 4+ 2+ 4+ 4+ 2+ 72 Alsevers 4+ 2+ 4+ 3+ Cellstab ™ 4+ 4+ 3+/2+ 4+ 4+ 3+ 87 Alsevers Cellstab ™ 4+ 4+/3+ 1+ 4+ 4+/3+ 2+/1+ 116 Alsevers Cellstab ™ 4+ 3+ 0  4+ 4+/3+ 1+

Example 9 Red Blood Cell Transformation with H-antigen Synthetic Glycolipids

The water-soluble synthetic glycolipids designated H_(tri-sp-Ad-DOPE (VII), H) _(di)-sp-Ad-DOPE (VIII) and Galβ-sp-Ad-DOPE (IX) were prepared according to the method described in Example 1 with necessary modifications.

Washed packed mouse RBCs (3 parts by volume) and the synthetic glycolipid solutions (1 part by volume of varying concentrations) were added to an eppendorf tube. The tube was incubated in a 37° C. waterbath for one hour, mixing every 15 minutes. The transformed RBCs were washed 3× with PBS and then suspended in Cellstab™ at the appropriate concentration for serology testing.

Tube serology and Diamed gel-card results for RBCs transformed with the different synthetic glycolipids are presented in Table 46. The results show that three sugars (H_(tri)) are required for detection by anti-H IgM, at least by the reagent used.

TABLE 45 Antisera used for results presented in Table 46. Antisera Manufacturer Batch Anti-H IgM Japanese Red Cross HIRO-75 UEA Lorne Laboratories 11549E D.O.E. 06.2004 Bio-UEA EY Labs 201105-2

TABLE 46 Comparison of transformation of RBCs using H-antigen synthetic glycolipids with different glycotopes made to different concentrations (n.d.—not determined). H Antisera UEA Conc IgM Tube Tube Bio-UEA Synthetic mg/mL Tube Diamed T0 T20 Tube H_(tri)-sp- 1 n.d. n.d. 2+ n.d. 2+ Ad-DOPE 0.25  4+ 3+ n.d. n.d. 1+ (VII) 0.1  3+ 2+ n.d. n.d. n.d. 0.05  1+ 0  n.d. n.d. n.d. 0.01 0 0  n.d. n.d. n.d. H_(di)-sp- 0.25 0 n.d. n.d. n.d. n.d. Ad-DOPE 0.1 0 n.d. n.d. n.d. n.d. (VIII) 0.05 0 n.d. n.d. n.d. n.d. 0.01 0 n.d. n.d. n.d. n.d. Galβ-sp- 0.25 0 n.d. n.d. n.d. n.d. Ad-DOPE 0.1 0 n.d. n.d. n.d. n.d. (IX) 0.05 0 n.d. n.d. n.d. n.d. 0.01 0 n.d. n.d. n.d. n.d. Human O —  4+ n.d. 1+ 2/3+ 4+ cells Incubated — 0 n.d. 0  0 n.d. control Bench — 0 n.d. n.d. n.d. n.d. control

Example 10 Insertion of H_(di)-sp-Ad-DOPE (VIII) and Galβ-sp-Ad-DOPE (IX) Synthetic Glycolipids into Murine Red Blood Cells

The water soluble synthetic glycolipids designated H_(di)-sp-Ad-DOPE (VIII) and Galβsp-Ad-DOPE (IX) were prepared according to the method described in Example 1 with necessary modifications.

Murine RBCs were washed 3× in 1× PBS. 30 μl of packed RBCs were combined with 30 μl of H_(ai)-sp-Ad-DOPE (VIII), and 30 μl of packed RBCs were combined with 30 μl Galβ-sp-Ad-DOPE (IX), respectively. Both synthetic molecule constructs were at a concentration of 1.0 mg/ml. 30 μof 1× PBS was added to 30 μl of packed RBCs to act as the control group. Cells were incubated for 90 minutes in a 37° C. shaking water-bath. RBCs were washed 3× in 1× PBS.

Three groups of packed RBCs were incubated with an equal volume of lectin UEA-1 for 30 minutes at room temperature. The lectin was prepared in 1× PBS at a concentration of 0.1 mg/ml. 50 μl of a 3% cell suspension was spun for 15 seconds in an Immunofuge at low speed. Results were read by tube serology. The results are presented in Table 48. The results show that neither anti-H IgM nor UEA-1 detects two sugars (H_(di) ).

TABLE 47 Antisera used for results presented in Table 48. Antisera Manufacturer Batch Biotest anti-H Biotest AG UEA EY Labs 201105-2

TABLE 48 Murine RBCs transformed with Galβ-sp-Ad-DOPE or Hdi-sp-Ad-DOPE, assessed by agglutination. Cell Type Inserted Molecule UEA-1 Mouse IgM^(H) Murine RBC Galβ (1 mg/ml) 0 n.d. Murine RBC H_(di) (1 mg/ml) 0 0 Murine RBC Control (PBS) 0 0 Human RBC Control (PBS) 4+ 3+ Abbreviations: n.d. Not determined

Example 11 Preparation of Sensitivity Controls

The synthetic glycolipids of the invention may be used in the preparation of “sensitivity controls” (also referred to as “quality control cells”, “serology controls”, or “process controls”) as described in the specification accompanying international application no. PCT/NZ02/00214 (WO 03/034074). The synthetic glycolipids provide the advantage that the transformation of the RBCs may be achieved at reduced temperatures.

RBC Transformation Solutions

Two stock solutions are used:

-   -   Solution 1: 1mg/mL A_(tri)-sp-Ad-DOPE (I) suspended in         Celpresolm solution.     -   Solution 2: 5mg/mL B_(tri)-sp-Ad-DOPE (VI) suspended in         Celpresolm solution.

Glycolipids are manufactured in a white dry powder. Glycolipids in this form (enclosed in a sealed container under a controlled temperature) are stable for an indefinite period of time. The glycolipids are suspended in solution (e.g. Celpresol™) by weight in order to formulate the transformation solutions.

Once the transformation solutions are received at CSL, they are filtered (through a MILLEX®-GV 0.22μ filter unit) under aseptic conditions.

Processing of RBCs

RBC donations are processed using a continuous flow centrifuge washer under aseptic conditions. RBC donations are washed in buffered saline followed by Celpresol™ solution. The PCV of the RBC donations is measured on a Beckman Coulter AcT Diff analyser. The donations are then adjusted to a packed cell volume (PCV) of 50% with the addition of Celpresol™.

Transformation of RBCs to Provide “Weak AB Cells”

RBCs are washed in buffered saline and Celpresol™. The cells are suspended in Celpresol™ solution to a PCV of>50%. The PCV of red cells is measured using a Beckman Coulter AcT Diff. The mass of the red cell solution is weighed.

The amount of A_(tri)-sp-Ad-DOPE (I), B_(tri)-sp-Ad-DOPE (VI) and Celpresol™ for transformation is calculated using the following equations:

$a = \frac{P \times F}{S}$ $b = \frac{P \times F}{S}$ c = P − (1 − P) − a − b

where

-   -   a=amount of A_(tri)-sp-Ad-DOPE (I) to be added per 1 mL of red         cells (mL)     -   b=amount of B_(tri)-sp-Ad-DOPE (VI) to be added per 1 mL of red         cells (mL)     -   c=amount of Celpresol™ to be added per 1 mL of red cells (mL) to         dilute cells to 50% PCV     -   P=PCV of red cell solution     -   F=Final desired concentration of glycolipid     -   S=Concentration of stock glycolipid solution

To determine the amount of glycolipid and Celpresol™ to add to a bulk sample of red cells, multiply each of a, b and c by the red cell volume. Add A_(tri)-sp-Ad-DOPE (I), B_(tri)-sp-Ad-DOPE (VI) and Celpresol™ to the red cell bulk sample aseptically.

Incubate the sample for 3 hours at 20° C. under controlled temperature conditions and constant gentle agitation. At the end of the 3 hour period, aseptically remove a sample of red cells and test the sample to confirm transformation of the RBCs. Perform blood grouping using tube, tile and column agglutination technology (CAT) techniques.

Incubate the red cell sample for 3 hours at 2-8° C. under controlled temperature conditions and constant gentle agitation for 18 hours. At the end of the 3 hour period, aseptically remove a sample of red cells and test the sample to confirm transformation of the red cells. Perform blood grouping using tube, tile and CAT techniques.

Wash the transformed red cells using a continuous flow centrifuge method, under aseptic conditions using Celpresol™ solution. Measure the PCV of the washed red cells and adjust to 50% PCV by the addition of CelpresolTM solution.

Formulation and Dispensing

Aseptically combine a volume of the transformed RBCs with a volume of simulated plasma diluent (SPD). The plasma may contain monoclonal and polyclonal antibodies. Antibodies are selected according to the desired characteristics of the sensitivity controls. The plasma may additionally contain tartrazine and bovine serum albumin.

Blood grouping and antibody screening is performed on the bulk samples using tube, tile and CAT techniques. The transformed RBC-SPD blend is then aseptically dispensed into BD Vacutainer tubes and the tubes labelled accordingly.

Validation Testing

Weak AB cells produced by the use of synthetic glycolipids (designated A_(w)B_(w) in Tables 51 to 53) were used to validate a range of testing platforms in parallel with naturally occurring weak A, weak B and weak AB cells.

TABLE 49 Reagents and cards used in validation testing. Method Reagent Tube Epiclone Tile Epiclone Ref Manufacturer and type Batch Expiry CAT 1 OCD BioVue ABD/Rev ABR528A 16.06.05 CAT 2 OCD BioVue ABD/Rev ABR521A 06.05.06 CAT 3 OCD BioVue ABD/ABD ACC255A 24.05.05 CAT 4 Diamed ID-MTS 50092.10.02 Apr-05 CAT 5 Diamed ID-MTS Donor typing 51051.05.04 Mar-05 CAT 6 Diamed ID-MTS Recipient typing 50053.07.02 Apr-05 CAT 7 Diamed ID-MTS Cord typing 50961.08.03 Jul-05

TABLE 50 Testing platform methodology for validation testing. Tile 1 drop 3% cells, 2 drops reagent, 15 min @ RT in moist chamber. Tube 2 drops @ RT, 10 min. ID-MTS As per manufacturers instructions using Dil-2. BioVue As per manufacturers instructions using 0.8% RCD.

TABLE 51 Validation results across all methods against anti-A. Testing platform Cell Type Tube Tile CAT 1 CAT 2 CAT 3 CAT 4 CAT 5 CAT 6 CAT 7 1 A_(x) w+ 0 2+ 1+ 0 0 0 0 2 A_(x) w+ 0 2+ 2+ 0 0 0 0 3 A₁B 4+  4+ 4+  4+ 4+  4+  4+  4+  4+ 4 A_(x) w+ 0 2+ 2+ 0 0 0 0 5 A₂B 3+  3+ 4+ 3+  3+  1+  2+  3+ 6 A_(x) w+ 0 2+ 2+ 0 0 0 0 7 A_(x) 1+ 0 2+ 2+ 0 0 0 0 8 A_(x) w+ 0 2+ 2+ 0 0 0 0 9 A_(x) 0  0 1+ 1+ 0 0 0 0 10 A_(x) w+ 0 2+ 2+ 0 0 0 0 11 A₃ 4+  4+ 4+ 3+  3+  1+  1+  3+ 12 A₃B 3+  3+ 3+ 3+  2+  w+  w+  2+ 13 B₃ 0  0 0  0 0  0 0 0 0 14 B₃ 0  0 0  0 0  0 0 0 0 15 A_(w)B_(w) 2+  2+ 2+  2+ 2+ 0 0 0 0

TABLE 52 Validation results across all methods against anti-B. Testing platform Cell Type Tube Tile CAT 1 CAT 2 CAT 3 CAT 4 CAT 5 CAT 6 CAT 7 1 A_(x) 0 0 0 0 0 0 0 0 2 A_(x) 0 0 0 0 0 0 0 0 3 A₁B  4+  4+  4+ 4+  4+  4+  3+  3+  4+ 4 A_(x) 0 0 0 0 0 0 0 0 5 A₂B  4+  4+  4+  4+  4+  3+  3+  4+ 6 A_(x) 0 0 0 0 0 0 0 0 7 A_(x) 0 0 0 0 0 0 0 0 8 A_(x) 0 0 0 0 0 0 0 0 9 A_(x) 0 0 0 0 0 0 0 0 10 A_(x) 0 0 0 0 0 0 0 0 11 A₃ 0 0 0 0 0 0 0 0 12 A₃B  4+  4+  4+  4+  4+  4+  4+  4+ 13 B₃  2+  2+  3+ 2+  2+  2+  2+  2+  2+ 14 B₃  2+  2+  2+ 2+  2+  2+  1+  1+  2+ 15 A_(w)B_(w)  3+  3+  1+ 1+  1+ 0 0 0 0

TABLE 53 Validation results across all methods against anti-AB. Testing platform Cell Type Tube Tile CAT 1 CAT 2 CAT 3 CAT 4 CAT 5 CAT 6 CAT 7 1 A_(x) 3+ 2+ 2+ 2 A_(x) 4+ 2+ 3+ 3 A₁B 4+ 4+ 4+ 4 A_(x) 3+ 2+ 3+ 5 A₂B 4+ 4+ 4+ 6 A_(x) 4+ 4+ 3+ 7 A_(x) 4+ 4+ 3+ 8 A_(x) 3+ 4+ 3+ 9 A_(x) 4+ 2+ 2+ 10 A_(x) 3+ 4+ 3+ 11 A₃ 4+ 4+ 4+ 12 A₃B 4+ 4+ 4+ 13 B₃ 2+ 2+ 2+ 14 B₃ 2+ 2+ 2+ 15 A_(w)B_(w) 3+ 3+ 3+

Example 12 Attachment of Modified Embryos to Transformed Endometrial Cells

The ability to effect qualitative and quantitative differences in the cell surface antigens expressed by cell types other than RBCs was investigated. The ability to enhance the adhesion of embryos to endometrial cells was adopted as a model system.

The synthetic molecules may be used as synthetic membrane anchors and/or synthetic molecule constructs. Therefore, they may also be employed in the method of enhancing embryo implantation as described in international patent application no PCT/NZ2003/000059 (published as WO 03/087346) which is incorporated by reference.

-   -   Endometrial Cell Transformation

Insertion of Water Soluble Synthetic Molecule Construct

A single cell suspension of endometrial epithelial cells was prepared. The endometrial cells were washed 3× by resuspending in CMF HBSS and centrifuging at 2000 rpm for 3 minutes. The washed cell preparation was resuspended in 50 μl of M2.

Micro-centrifuge tubes each containing a 50μ1 solution of 5M/ml endometrial cells were prepared. To separate tubes of endometrial cells 50 μ1 of synthetic glycolipids A_(tri)-sp-Ad-DOPE (I) or B_(tri)-sp-Ad-DOPE A (VI), or 50 μ1 M2 were added to the control cells. The cells were incubated for 90 minutes at 37° C. on a mixer. The endometrial cells were washed 3× by resuspending in CMF HBSS media and centrifuging at 2000 rpm for 3 minutes. The washed cell preparation was resuspended in 50 μl of M2.

Test For Insertion Using Fluorescent Probe:

50 μl of corresponding primary murine monoclonal antibody was added to each tube. Each tube was incubated at room temperature for 10 minutes. Cells were washed 3× in M2 media. 10 μl of mouse anti-IgG FITC was added to each tube. Tubes were incubated at room temperature in dark conditions for 10 minutes. Endometrial cells were mounted on glass slides and viewed under a fluorescence microscope.

Test for Direct Agglutination:

5 μl of each group of cells was placed onto separate microscope slides. To each 5 μl drop of cells 5 μl of a corresponding antibody was added. The cells were gently mixed on the slide for 2 minutes. Agglutination was visualised under the microscope. The results are presented in Table 55.

TABLE 54 Antisera used for results presented in Table 55. Antisera Manufacturer Bioclone anti-A Ortho Diagnostics 01102 D.O.M. 16.05.02 Bioclone anti-B Ortho Diagnostics Developmental reagent

TABLE 55 Endometrial cells transformed with A_(tri)-sp-Ad-DOPE (I) or B_(tri)-sp-Ad-DOPE A (VI), as visualised using fluorescence. Fluorescence Agglutination score after reaction by Inserted 1° incubation with microscopic Cell Type Antigen antibody IgFITC Probe visualisation Endometrial A_(tri)-sp-Ad- Anti-A 4+ 4+ cells DOPE (I) Bioclone (1 mg/ml) Endometrial B_(tri)-sp-Ad- Anti-B 1+ 3+ cells DOPE (VI) Bioclone (1 mg/ml) Endometrial Control (M2 Anti-A 0 0 cells media) Bioclone

-   -   Embryo Modification

Insertion of Water Soluble Synthetic Molecule Construct:

The embryo zona pellucida was removed by treating embryos with 0.5% pronase in a 37° C. oven for 6 minutes or until all zonas were removed. Micro-drops were prepared by adding 5μl of synthetic glycolipid A_(tri)-sp-Ad-DOPE (I) or B_(tri)-sp-Ad-DOPE (VI), at a concentration of 1 mg/mlL to a 45 μl drop of M2 media overlaid with mineral oil. All embryo groups were incubated in the 50 μl micro-drops for 1 hour at 37° C. Embryos from experimental and control groups were washed 3× with M2 media.

Test for Insertion:

Embryos from experimental and control groups were placed into a micro-drop of corresponding antibody and incubated for 30 min at 37° C. Embryos from experimental and control groups were washed 3× with M2 media.

Embryos from all experimental and control groups were placed into micro-drops of anti-mouse Ig FITC (1:50 dilution anti-mouse Ig FITC in M2) and incubated for 30 min at 37° C. Embryos from experimental and control groups were washed 3× with M2 media. Embryos were mounted on microscope slides in a 5 μl drop of M2 and the drops overlaid with oil.

The slides were viewed under a fluorescence microscope. Results are presented in Tables 56 and 57. The negative result for transformation with B_(tri)-sp-Ad-DOPE (VI) is attributed to a lack of 1° antibody sensitivity.

TABLE 56 Embryos transformed with Atri-sp-Ad-DOPE (I) as visualised using fluorescence. Fluorescence Embryo score after Morphology Cell Inserted 1° incubation with 24 hr post Type Antigen antibody IgFITC Probe insertion Embryos A_(tri)-sp-Ad- Anti-A 2+/1+ Appeared DOPE (I) Bioclone viable Embryos Control Anti-A 0 Appeared Bioclone viable

TABLE 57 Embryos transformed with A_(tri)-sp-Ad-DOPE (I) or B_(tri)-sp-Ad- DOPE (VI), as visualised using fluorescence. Fluorescence Embryo score after Morphology Cell Inserted 1° incubation with 24 hr post Type Antigen antibody IgFITC Probe insertion Embryos A_(tri)-sp-Ad- Anti-A 2+ n.d. DOPE (I) Bioclone Embryos B_(tri)-sp-Ad- Anti-B 0 n.d. DOPE (VI) Bioclone Embryos Control Anti-A 0 n.d. (M2 media) Bioclone

Enhanced Attachment Transformed Endometrial Cells to Modified Embryos

Modified embryos (BioG-Avidin-BioIgG^(B) and BioG-Avidin-BioIgM^(A)) were prepared in accordance with the methods described in the specification accompanying the international application no. PCT/NZ03/00059 (published as W003/087346).

Two concave glass slides were prepared, one with two wells of synthetic glycolipid A_(tri)-sp-Ad-DOPE (I) inserted endometrial cells and the other with two wells of synthetic glycolipid B_(tri)-sp-Ad-DOPE (VI) inserted endometrial cells.

The two groups of embryos were transferred to each of the concave glass slides:

Slide 1 A_(tri)/IgG^(B) embryos

-   -   A_(tri)/IgM^(A) embryos

Slide 2 B_(tri)/IgG^(B) embryos

-   -   B_(tri)/IgM^(A) embryos

The embryos were surrounded with endometrial cells. The wells were covered with mineral oil and incubated for 15 minutes at 37° C. Using a wide bore handling pipette each group of embryos were carefully transferred to a fresh drop of M2 media. The embryos were gently washed. The embryos were gently transferred into 2 μL of M2 media on a marked microscope slide. Each drop was overlaid with mineral oil

Under a central plane of focus on an Olympus microscope the number of endometrial cells adhered to the embryos in each group was assessed. The number of cells adhered to each embryo was recorded. Results are presented in Table 58.

TABLE 58 Endometrial cells transformed with A_(tri)-sp-Ad-DOPE (I) or B_(tri)-sp-Ad-DOPE (VI), and embryos modified with BioG-Avidin-BioIgGB or BioG-Avidin-BioIgMA. Assessment by attachment of endometrial cells to embryos. Average number of endometrial Transformed Modified cells attached Cell Type endometrial cells embryos to modified embryos Endometrial A_(tri)-sp-Ad-DOPE BioG- 2.3 cells (I) Avidin- BioIgG^(B) BioG- 7.25 Avidin- BioIgM^(A) Endometrial B_(tri)-sp-Ad-DOPE BioG- 6.7 cells (VI) Avidin- BioIgG^(B) BioG- 3.4 Avidin- BioIgM^(A)

Example 13

Association of KODE-Fluorescein (I) with Cell Membranes

KODE-fluorescein (I) readily associates with the membrane of red blood cells. Insertion of the molecule is observed when dispersions of the molecule at concentrations greater than 0.1 mg/ml are contacted with suspensions of the red blood cells.

A medium to strongly fluorescing cell was considered to indicate a uniform distribution of the molecule across the cell membrane (FIG. 20). The incorporation and distribution appears to be stable for a period of at least 40 days when cells are stored in the dark.

Where in the foregoing description reference has been made to integers or components having known equivalents then such equivalents are herein incorporated as if individually set forth.

Although the invention has been described by way of example and with reference to possible embodiments thereof it is to be appreciated that improvements and/or modification may be made thereto without departing from the scope or spirit of the invention.

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1) A construct of the structure F-S₁-S₂-L where: F-S₁ is an aminoalkylglycoside comprising the terminal sugars Galα1-3Galβ and S₁ is 2-aminoethyl, 3-aminopropyl, 4-aminobutyl or 5-aminopentyl; S₂ is —CO(CH₂)₂CO—, —CO(CH₂)₃CO—, —CO(CH₂)₄CO— or —CO(CH₂)₅CO—; and L is phosphatidylethanolamine. 2) The construct of claim 1 where the aminoalkylglycoside is an aminoalkyltrisaccharide. 3) The construct of claim 2 where S₁ is 3-aminopropyl. 4) The construct of claim 3 where S₂ is —CO(CH₂)₄CO—. 5) The construct of claim 4 where L is dioleoylphosphatidylethanolamine. 